CN104101589A - Detecting method used for detecting permeability value of cell resting membrane potential and sodium potassium ion based on TMRM - Google Patents

Abstract

The invention discloses a detecting method used for detecting the permeability value of cell resting membrane potential and sodium potassium ions based on TMRM (Tetramethylrhodamine Methyl Ester). The detecting method is characterized in that the traditional cell resting membrane potential detecting method based on TMRM is improved; C 17.2 nerve stem cells are used as models, and the nonspecific adsorption of the TMRM in a cell nucleus section is accurately demarcated by adopting a binding site method, so that the cell resting membrane potential can be calculated accurately; based on the method, sodium concentration in cells is detected by sodium fluorochrome CoroNaGree, and the permeability value of the sodium potassium ions can be further calculated, so that the detecting method, with higher accuracy, used for detecting the permeability value of the non-invasive cell resting membrane potential and the sodium potassium ions, can be established. According to the invention, the permeability value of the cell resting membrane potential and the sodium potassium ions can be detected more accurately for planar cells and three-dimensional cells.

Description

Cell resting membrane electric potential based on TMRM and sodium potassium ion permeability compare value detection method

Technical field

The invention belongs to biological technical field, relate to the detection method of a kind of cell membrane potential and sodium potassium ion permeability ratio.

Background technology

Under quiescent condition, uneven distribution and the permeability difference of different ions on cell membrane of the inside and outside both sides of cell membrane ion have formed cell resting membrane electric potential (resting membrane potential, RMP), it is the basis that all living things electricity produces and changes, and many functional memebrane proteins are had to regulating action as ion channel (ion channels), transporter (transporters), sodium potassium pump (pumps), enzyme (enzymes) etc.The adjusting of the change cell membrane functional protein of RMP is the key that electrical stimulation signal changes into intracellular signal transduction, and and then affects the propagation of cell, differentiation, apoptosis etc.Therefore accurately detect RMP significant.Because cell membrane is relatively little to the permeability of chlorion, so substantially determined the size of cell resting membrane electric potential by the CONCENTRATION DISTRIBUTION of the known sodium potassium ion of Goldman – Hodgkin – Katz equation and their permeability ratio on cell membrane.

Current detecting cell resting membrane electric potential is mainly by patch-clamp and two kinds of methods of electromotive force fluorescent dye.Patch-clamp method has damage to a certain degree to cell owing to detecting, and has changed intracellular actual ions concentration ratio, is clamped down on cell easily dead, is more suitable for the electric physiological behavior of cell passage albumen is detected.Electromotive force fluorescent dye method is by carrying out indicator cells resting membrane electric potential by the fluorescence intensity of the charged dyestuff of Potential Distributing, there is Noninvasive, more easily obtain the real cell resting membrane electric potential of cell, the in situ detection that also can cannot operate for some patch-clamp methods simultaneously, detect flux also higher, but there is the not high enough problem of accuracy of detection in the method.Tetramethyl rhodamine methyl esters (tetramethylrhodamine, methyl ester, TMRM) be a kind of red electromotive force fluorescent dye with monovalence positive charge, can freely pass through cell membrane, in film both sides, form this special balanced distribution of stable energy, toxicity to cell is relatively little, is considered to a kind of desirable detection cell resting membrane electric potential and the fluorescent dye of mitochondrial membrane potential.Yet while utilizing at present TMRM to carry out the detection of cell resting membrane electric potential, to the demarcation of the non-specific adsorption in its fluorescent particles He district, be to be based upon under the prerequisite that adsorption coefficient is constant, the non-specific adsorption of this and bibliographical information exists the conclusion of saturation history there are differences, thus precision when impact is demarcated cell resting membrane electric potential with TMRM.Therefore, need to demarcate TMRM He district non-specific adsorption, and then set up a kind of cell resting membrane electric potential detection method with degree of precision.

Sodium potassium ion permeability ratio P _na/Kas the key factor that affects cell resting membrane electric potential, be often used as an important indicator of research cellular electrophysiologicalsensor, especially in the research of ion channel function.Most of ionophorous protein on cell membrane is all specific a certain ion to be had to higher permeability, but it also has certain permeability to other ion, because sodium, potassium ion are the main kations inside and outside cell, so sodium potassium ion permeability ratio has decisive significance to the function of ionophorous protein.The research of most of sodium potassium ion permeability ratio all focuses on the channel protein responding fast in action potential.In recent years, the sodium potassium ion permeability ratio of the passage (leakage passage) that some bases under quiescent condition are activated is also studied, K2P passage for example, Kir passage and non-selective cationic channel (such as Nalcn, be considered to a kind of sodion and leak passage).K2P passage is considered to have the P that is less than 0.03 _na/K, the P of Kir passage _na/Kbe considered to be less than 0.04.These long-term open passages have determined the film potential value of quiescent condition together to the permeability of sodium potassium ion and sodium potassium pump to the transhipment of sodium potassium ion.Yet most of P _na/Kcalculating be all based on patch-clamp method, the limitation in view of aforementioned diaphragm clamping method exists, compares value detection method so need to set up a kind of sodium potassium ion permeability with the Noninvasive of good accuracy.

Summary of the invention

In view of this, one of object of the present invention is TMRM He district non-specific adsorption to demarcate, and then set up a kind of cell resting membrane electric potential detection method with degree of precision, two of object is on the basis of the set up cell resting membrane electric potential detection method with degree of precision, further calculate sodium potassium ion permeability ratio, and then set up a kind of sodium potassium ion permeability with the Noninvasive of degree of precision and compare value detection method.

After deliberation, the invention provides following technical scheme:

1. the cell resting membrane electric potential detection method based on TMRM, comprises the following steps:

The relation of B.TMRM fluorescence intensity and concentration is demarcated

TMRM is mixed with to the solution of a series of gradient concentrations, above-mentioned solution is carried out respectively to fluorescence intensity detection, by the TMRM fluorescence intensity recording and corresponding TMRM for concentration formula (1) carry out matching:

F＝k[TMRM ⁺] (1)

Wherein F is TMRM fluorescence intensity, [TMRM ⁺] be TMRM concentration; Try to achieve calibration coefficient k;

B. the non-specific adsorption of nuclear area TMRM is demarcated

First with potassium, penetrating dose of processing cell of sodion, make the complete depolarization of cell, the inside and outside free TMRM concentration of cell equates, with the TMRM solution of a series of gradient concentrations, distinguish incubated cell again, measure nuclear area TMRM fluorescence intensity, through type (1) calculates nuclear area TMRM total concentration, then through type (2) calculates the TMRM concentration of nuclear area non-specific binding:

$\left[{\mathrm{TMRM}}_i^{+}\right]=\left[{\mathrm{TMRM}}_B^{+}\right]+\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]---\left(2\right)$

[TMRM wherein _i ⁺] be nuclear area TMRM total concentration, [TMRM _b ⁺] be the TMRM concentration of nuclear area non-specific binding; for the free TMRM concentration in nuclear area, its value and the born of the same parents TMRM concentration of dissociating outward equates;

Then, by the free TMRM concentration of the TMRM concentration of the nuclear area non-specific binding calculating and nuclear area, with the model of two independent binding sites of an aglucon, be that formula (3) is carried out matching:

$\left[{\mathrm{TMRM}}_B^{+}\right]=\frac{\left[{\mathrm{TMRM}}_{B\max 1}^{+}\right]\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}{K_{D1}+\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}+\frac{\left[{\mathrm{TMRM}}_{B\max 2}^{+}\right]\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}{K_{D2}+\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}---\left(3\right)$

[TMRM wherein ⁺ _bmax1] and [TMRM ⁺ _bmax2] be respectively the TMRM concentration of maximum non-specific binding in two independent binding sites, K _d1and K _d2be respectively the dissociation constant of TMRM and two independent binding sites; Try to achieve [TMRM ⁺ _bmax1], [TMRM ⁺ _bmax2], K _d1and K _d2;

C. the cell resting membrane electric potential based on TMRM detects

With certain density TMRM solution incubated cell, measure the TMRM fluorescence intensity of nuclear area, through type (1) calculates nuclear area TMRM total concentration, and formula (3) substitution formula (2) is solved and obtained through type (4) calculates cell resting membrane electric potential again:

$V_m=-60\mathrm{lo}{g}_{10}\frac{\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}{\left[{\mathrm{TMRM}}_o^{+}\right]}---\left(4\right)$

V wherein _mfor cell resting membrane electric potential, [TMRM _o ⁺] be extracellular TMRM concentration.

Preferably, in steps A, be to contain 5mM KCl, 130mM NaCl, 1mM MgCl ₂, 2mM CaCl ₂with the 20mM HEPES damping fluid of 10mM glucose be solvent, TMRM is mixed with to concentration is respectively 5,50,100,250,500,1500,2500,3500,5000,10000, the solution of 50000nM.

Preferably, penetrating dose of potassium described in step B, sodion are respectively valinomycins and coban, first with containing 1 μ M valinomycins, 10 μ M cobans, 135mM KCl, 1mM MgCl ₂, 2mM CaCl ₂process cell with the 20mM HEPES damping fluid of 10mM glucose and make the complete depolarization of cell, then to contain 135mM KCl, 1mM MgCl ₂, 2mM CaCl ₂be respectively 500,1500,5000,10000 with the 20mM HEPES damping fluid of the 10mM glucose concentration that is solvent preparation, the TMRM solution incubated cell of 20000nM.

Preferably, described in step C, with certain density TMRM solution incubated cell, be to contain 5mM KCl, 130mM NaCl, 1mM MgCl ₂, 2mM CaCl ₂with the 20mM HEPES damping fluid of the 10mM glucose concentration that is solvent preparation the TMRM solution incubated cell that is 500nM.

2. the sodium potassium ion permeability based on TMRM, than value detection method, comprises the following steps:

The relation of A.TMRM fluorescence intensity and concentration is demarcated

TMRM is mixed with to the solution of a series of gradient concentrations, above-mentioned solution is carried out respectively to fluorescence intensity detection, by the TMRM fluorescence intensity recording and TMRM for concentration formula (1) carry out matching:

F＝k[TMRM ⁺] (1)

Wherein F is TMRM fluorescence intensity, [TMRM ⁺] be TMRM concentration; Try to achieve calibration coefficient k;

B. the non-specific adsorption of nuclear area TMRM is demarcated

First with potassium, penetrating dose of processing cell of sodion, make the complete depolarization of cell, the inside and outside free TMRM concentration of cell equates, with the TMRM solution of a series of gradient concentrations, distinguish incubated cell again, measure nuclear area TMRM fluorescence intensity, through type (1) calculates nuclear area TMRM total concentration, then through type (2) calculates the TMRM concentration of nuclear area non-specific binding:

$\left[{\mathrm{TMRM}}_i^{+}\right]=\left[{\mathrm{TMRM}}_B^{+}\right]+\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]---\left(2\right)$

[TMRM wherein _i ⁺] be nuclear area TMRM total concentration, [TMRM _b ⁺] be the TMRM concentration of nuclear area non-specific binding; for the free TMRM concentration in nuclear area, its value and the born of the same parents TMRM concentration of dissociating outward equates;

Then, by the free TMRM concentration of the TMRM concentration of the nuclear area non-specific binding calculating and nuclear area, with the model of two independent binding sites of an aglucon, be that formula (3) is carried out matching:

$\left[{\mathrm{TMRM}}_B^{+}\right]=\frac{\left[{\mathrm{TMRM}}_{B\max 1}^{+}\right]\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}{K_{D1}+\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}+\frac{\left[{\mathrm{TMRM}}_{B\max 2}^{+}\right]\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}{K_{D2}+\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}---\left(3\right)$

[TMRM wherein ⁺ _bmax1] and [TMRM ⁺ _bmax2] be respectively the TMRM concentration of maximum non-specific binding in two independent binding sites, K _d1and K _d2be respectively the dissociation constant of TMRM and two independent binding sites; Try to achieve [TMRM ⁺ _bmax1], [TMRM ⁺ _bmax2], K _d1and K _d2;

C. the cell resting membrane electric potential based on TMRM detects

With certain concentration TMRM solution incubated cell, measure the TMRM fluorescence intensity of nuclear area, through type (1) calculates nuclear area TMRM total concentration, and formula (3) substitution formula (2) is solved and obtained through type (4) calculates cell resting membrane electric potential again:

$V_m=-60\mathrm{lo}{g}_{10}\frac{\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}{\left[{\mathrm{TMRM}}_o^{+}\right]}---\left(4\right)$

V wherein _mfor cell resting membrane electric potential, [TMRM _o ⁺] be extracellular TMRM concentration;

D. in born of the same parents, Na ion concentration detects during gradient depolarization

First with potassium, penetrating dose of processing cell of sodion, make the inside and outside sodion balance of cell, cell is loaded to sodion fluorescent dye CoroNa Green simultaneously, with the sodion solution of a series of gradient concentrations, distinguish incubated cell again, measure cell fluorescence intensity, by fluorescence intensity and born of the same parents for Na ion concentration formula (5) carry out matching:

$\left[{\mathrm{Na}}_i^{+}\right]=\frac{K_d(F-F_{\min })}{F_{\max }-F}---\left(5\right)$

[Na wherein _i ⁺] be Na ion concentration in the born of the same parents under corresponding fluorescence intensity, F is the fluorescence intensity level under corresponding Na ion concentration; F _minfor not containing the fluorescence intensity level of sodion, by obtaining with the solution incubated cell that does not contain sodion; F _maxfor containing the fluorescence intensity level of saturated sodion, in cell, in the complete saturated situation of CoroNa Green, obtain by making with 1000mM sodion solution incubated cell; Try to achieve the dissociation constant K of sodion and CoroNa Green _d;

Then, detect as stated above the fluorescence intensity of cell in the gradient concentration potassium ion solution that adds CoroNa Green do not use potassium, penetrating dose of processing of sodion, the fluorescence intensity substitution formula (5) recording is calculated to Na ion concentration in born of the same parents, then by the outer potassium concentration of born of the same parents and born of the same parents for Na ion concentration formula (6) carry out matching:

$\left[{\mathrm{Na}}_i^{+}\right]=\mathrm{\Φ}\left(\right[K_o^{+}\left]\right)---\left(6\right)$

[K wherein _o ⁺] be the outer potassium concentrations of born of the same parents; Try to achieve Φ;

E. sodium potassium ion permeability ratio detects

Cell resting membrane electric potential can calculate with the Goldman equation of simplifying:

$V_m=-60\mathrm{lo}{g}_{10}\frac{\left[K_i^{+}\right]+P_{\mathrm{Na}/K}\left[{\mathrm{Na}}_i^{+}\right]}{\left[K_o^{+}\right]+P_{\mathrm{Na}/K}\left[{\mathrm{Na}}_o^{+}\right]}---\left(7\right)$

[K wherein _i ⁺] be potassium concentration in born of the same parents, [Na _o ⁺] be born of the same parents' extracellular sodium ion concentration; Corresponding different [K _o ⁺] [Na _i ⁺] can be obtained by formula (6); The in the situation that inside and outside cell, sodium potassium concentration summation being equal, V _mcan be by [K _o ⁺] represent:

$V_m=-60{\log }_{10}\frac{135-\mathrm{\Φ}\left(\right[K_o^{+}\left]\right)+P_{\mathrm{Na}/K}\mathrm{\Φ}\left(\right[K_o^{+}\left]\right)}{\left[K_o^{+}\right]+P_{\mathrm{Na}/K}(135-[K_o^{+}\left]\right)}---\left(8\right)$

Wherein, P _na/Kfor sodium potassium ion permeability ratio;

With the gradient concentration potassium ion solution incubated cell that contains finite concentration TMRM, measure the TMRM fluorescence intensity of nuclear area, through type (4) calculates corresponding cell resting membrane electric potential, then by cell resting membrane electric potential and born of the same parents outer for potassium concentration formula (8) carry out matching, can obtain P _na/K.

Preferably, in steps A, be to contain 5mM KCl, 130mM NaCl, 1mM MgCl ₂, 2mM CaCl ₂with the 20mM HEPES damping fluid of 10mM glucose be solvent, TMRM is mixed with to concentration is respectively 5,50,100,250,500,1500,2500,3500,5000,10000, the solution of 50000nM.

Preferably, penetrating dose of potassium described in step B, sodion are respectively valinomycins and coban, first with containing 1 μ M valinomycins, 10 μ M cobans, 135mM KCl, 1mM MgCl ₂, 2mM CaCl ₂process cell with the 20mM HEPES damping fluid of 10mM glucose and make the complete depolarization of cell, then to contain 135mM KCl, 1mM MgCl ₂, 2mM CaCl ₂be respectively 500,1500,5000,10000 with the 20mM HEPES damping fluid of the 10mM glucose concentration that is solvent preparation, the TMRM solution incubated cell of 20000nM.

Preferably, described in step C, with certain density TMRM solution incubated cell, be to contain 5mM KCl, 130mM NaCl, 1mM MgCl ₂, 2mM CaCl ₂with the 20mM HEPES damping fluid of the 10mM glucose concentration that is solvent preparation the TMRM solution incubated cell that is 500nM.

Preferably, penetrating dose of potassium described in step D, sodion are respectively valinomycins and coban, first with containing 1 μ M valinomycins, 10 μ M cobans, 5 μ M CoroNa Green, 5mM KCl, 130mM NaCl, 1mM MgCl ₂, 2mM CaCl ₂process cell with the 20mM HEPES damping fluid of 10mM glucose and make the sodion balance inside and outside cell, with concentration, be respectively again the sodion solution incubated cell of 10mM, 25mM, 55mM, 80mM, 100mM, 120mM, 130mM, in described sodion solution, also contain KCl, the 1mM MgCl of variable concentrations ₂, 2mM CaCl ₂, 10mM glucose and 20mM HEPES, wherein kalium ion and natrium ion concentration summation is 135mM.

Preferred, the gradient concentration potassium ion solution that adds CoroNa Green described in step D is to contain that 5 μ M CoroNa Green and potassium concentration are respectively 5,7.5,15,35,55,80, the solution of 130mM, also contains NaCl, the 1mM MgCl of variable concentrations in described solution ₂, 2mM CaCl ₂, 10mM glucose and 20mM HEPES, wherein kalium ion and natrium ion concentration summation is 135mM.

Beneficial effect of the present invention is: the present invention improves traditional cell resting membrane electric potential detection method based on TMRM, take C17.2 neural stem cell as model, by the method in double combination site, demarcated comparatively accurately the TMRM non-specific adsorption of nuclear area, improved the precision while calculating cell resting membrane electric potential, and on the method basis, by sodion fluorescent dye CoroNa Green, Na ion concentration in born of the same parents is detected, further calculated sodium potassium ion permeability ratio, thereby set up a kind of cell resting membrane electric potential and sodium potassium ion permeability with the Noninvasive of degree of precision and compared value detection method.The method can be carried out cell resting membrane electric potential and the detection of sodium potassium ion permeability ratio comparatively accurately to the cell of plane cell and three dimensional growth.

Accompanying drawing explanation

In order to make object of the present invention, technical scheme and beneficial effect clearer, the invention provides following accompanying drawing and describe:

The fluorescence intensity of Fig. 1, TMRM and the graph of a relation of concentration and the distribution situation figure of TMRM fluorescence inside and outside C17.2 cell, wherein a is the result that the TMRM solution of variable concentrations is carried out to the scanning of laser co-focusing layer, the enlarged drawing that illustration is boxed area; B is C17.2 cell design sketch after TMRM dyeing; C be in b figure a to the change in fluorescence figure of b;

Fig. 2, the RMP based on TMRM detect and patch-clamp proof diagram, the Scatchard figure (n=26) that wherein a obtains after matching for utilizing equation (2) to carry out, figure Zhong “ ﹍ " with “ ﹎ " dotted line represents respectively two independent binding sites in equation, the graph of a relation (n=26) of the TMRM concentration that illustration is non-specific adsorption and free TMRM concentration; B adopts respectively the inventive method and patch-clamp method to detect the result of C17.2 cell RMP after gradient depolarization;

Fig. 3, P _na/Kdetection figure, wherein a is CoroNa Green calibration curve (n=24), illustration is CoroNa Color figure; The graph of a relation (n=21) of the interior Na ion concentration of born of the same parents and the outer potassium concentration of born of the same parents when b is gradient depolarization; C is 5～135mM K ⁺rMP in HBS solution and P thereof _na/Kthe matching (solid black lines) and the two kinds of Nernst curves (n=17) that calculate; D is 5～35mM K ⁺p in HBS solution _na/K(n=17) figure;

RMP and the P in the different propagation of Fig. 4, cell period _na/Kdetect figure, the cultivation cell RMP of 1,2, the 3 days figure that wherein a detects for adopting respectively the inventive method and patch-clamp method; B is for cultivating the interior Na ion concentration figure of cell born of the same parents of 1,2,3 day; The cultivation cell P of 1,2,3 day of c for adopting respectively the inventive method and patch-clamp method to detect _na/K(* represents p<0.001) figure;

RMP and the P of the cell of Fig. 5, the growth of three-dimensional aggregation _na/Kdetect figure, wherein a and b are respectively cell and differ micrograph and TMRM fluorogram in recessed " 80-40 " is small; C and d are respectively RMP and the P of cultured cell in small recessed and plane _na/K(* represents p<0.001) figure;

Fig. 6, the inventive method and the existing methodical comparison diagram based on TMRM, wherein a figure is to having added 5～130mM K of valinomycins ⁺fluorescence data by several different methods, calculate its RMP; B figure is to 5～130mM K ⁺fluorescence data by several different methods, calculate its RMP; C figure is to 5～130mM K ⁺fluorescence data by several different methods, calculate its P _na/K(n=17).

Embodiment

Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in preferred embodiment, conventionally according to normal condition, or the condition of advising according to reagent manufacturer is carried out.

One, cell is cultivated

The present embodiment adopts C17.2 neural stem cell to study.Because neural stem cell is considered to have more K2P and Kir expresses, for example, in the neural stem cell ball that Kir2.4, Kir4.1, Kir6.1, the Kir7.1 in TASK1, the TASK3 in K2P family, TREK1 HeKir family cultivates in vitro, there is expression, and there is not yet in bibliographical information neural stem cell, there is the non-selective cationic channel of the sustained activation such as Nalcn to express, ion permeability on this explanation neural stem cell film mainly leaks passage by potassium to be provided, so C17.2 is the cell that a desirable research potassium leaks passage sodium potassium ion permeability ratio., because neural stem cell has more negative RMP (lower than-70mV), can in wider potential change amplitude, to method to be measured, verify, so neural stem cell is also by TMRM method, to detect the ideal model of RMP meanwhile.

The cultural method of C17.2 neural stem cell is as follows: sterilized 25mm circular lid slide is put to 35mm double dish bottom, on it with 1 * 10 ⁵the density inoculation C17.2 neural stem cell of/mL, adopt and add 10% hyclone (GIBCO, USA), 100U/mL penicillin and 100 μ g/mL streptomycin sulphate (Sigma, USA) DMEM/F12 (1:1) basal medium (GIBCO, USA), put 37 ℃, 95% humidity and 5%CO ₂incubator in carry out amplification cultivation.

Two, the demarcation of TMRM

First the relation of the fluorescence intensity of TMRM and concentration is demarcated.Concrete grammar is as follows: 5mM KCl, 130mM NaCl, 1mM MgCl are added in preparation ₂, 2mM CaCl ₂(hereafter is 5mM K with the 20mM HEPES damping fluid of 10mM glucose ⁺hBS solution, described HBS solution is except special instruction herein, and potassium, Na ion concentration summation are 135mM, other constituent concentration is constant).With this 5mM K ⁺hBS solution is solvent, TMRM is mixed with to concentration is respectively 5,50,100,250,500,1500,2500,3500,5000,10000, the solution of 50000nM.The TMRM solution of variable concentrations is put respectively in the 1mL stainless steel double dish that includes 25mm circular lid slide, at laser confocal microscope (LSM510META, ZEISS, Germany) on 37 ℃ of warm tables, hatch after 25min, adopt the logical filter disc of the He-Ne Lasers of 543nm and the band of 560-615nm to carry out laser confocal imaging.First take 5mM K ⁺the background fluorescence figure of HBS solution, by concentration, from low to high TMRM solution is detected again, from glass-based bottom, upwards sweep 10 layers, every layer of 1 μ m, obtain fluorescence picture, utilize AXIOVISION REL.4.8 (ZEISS, Germany) software to carry out the analysis of fluorescence gray-scale value, the typical curve of drawing TMRM fluorescence intensity and concentration, obtains following equation:

F＝k[TMRM ⁺] (1)

Wherein F is TMRM fluorescence intensity, [TMRM ⁺] be TMRM concentration, k is calibration coefficient.

The result of a for the TMRM solution of variable concentrations is carried out to the scanning of laser co-focusing layer in Fig. 1, discovery is in the region apart from more than substrate of glass 1 μ m, basic and the concentration of the fluorescence intensity of TMRM solution is linear relationship completely, to carrying out matching apart from the data of substrate of glass 2-9 μ m, the k value of trying to achieve in equation (1) is 0.446.In experiment, also find, at the glass bottom (apart from substrate of glass 0 μ m), locate, the fluorescence intensity of TMRM solution is a little more than the fluorescent value away from bottom, this is perhaps with 1 valency positive charge because of TMRM, have the cause of electric charge absorption behavior with the impurity in glass, and the phenomenon that this fluorescence increases can ignored substantially apart from region more than substrate of glass 1 μ m.

In Fig. 1, b and c have shown the distribution situation of TMRM fluorescence inside and outside cell.

Three, the RMP based on TMRM detects and patch-clamp checking

1, the non-specific adsorption of TMRM is demarcated and the detection of the RMP based on TMRM

Owing to there being the band TV university molecules such as a large amount of protein, nucleic acid, therefore there is non-specific adsorption to TMRM in nuclear area, and then the assessment of impact to endocellular liberation TMRM concentration.Researcher Leow thinks that the TMRM of non-specific adsorption of nuclear area and free TMRM are a fixing ratio, but it is considered herein that the rising along with free TMRM concentration, the TMRM of non-specific adsorption is saturated by region, although this phenomenon is not obvious during in low concentration at free TMRM, but precision when it also detects RMP by impact with TMRM, for the more negative cell of current potential, because TMRM relative concentration in its born of the same parents is higher, above-mentioned impact will be more remarkable, therefore the non-specific adsorption of nuclear area be demarcated particularly important.

Valinomycins (valinomycin) and coban (monensin) are respectively potassium, sodion penetrating doses, and both use together and are considered to make the complete depolarization of cell to 0 current potential.The present invention adds 1 μ M valinomycins and 10 μ M cobans to 135mM K ⁺in HBS solution, make the complete depolarization of cell, it is-0.59 ± 1.34 mV (n=6) that patch-clamp detects potential value, substantially in 0 potential state, the inside and outside free TMRM concentration of cell equates, the TMRM solution that then adds variable concentrations outward born of the same parents is demarcated born of the same parents inner core region non-specific adsorption.

Concrete grammar is as follows: C17.2 cell is cultivated 3 days on 25mm circular lid slide, put in stainless steel double dish, with the 135mM K that adds 1 μ M valinomycins and 10 μ M cobans ⁺hBS solution is hatched after 1 hour in 37 ℃, puts on 37 ℃ of warm tables of laser confocal microscope, and the visual field immobilizes after being adjusted to the position with suitable cell density, respectively with adding 500,1500,5000,10000, the 135mM K of 20000nM TMRM ⁺hBS solution is hatched 25min, then the same visual field is taken, obtain apart from the fluorogram at substrate of glass 3 μ m places, utilize AXIOVISION software to carry out the analysis of fluorescence gray-scale value to the same nuclear area of every figure, by equation (1), calculate nuclear area TMRM total concentration, then through type (2) calculates the TMRM concentration of nuclear area non-specific binding:

$\left[{\mathrm{TMRM}}_i^{+}\right]=\left[{\mathrm{TMRM}}_B^{+}\right]+\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]---\left(2\right)$

[TMRM wherein _i ⁺] be nuclear area TMRM total concentration, [TMRM _b ⁺] be the TMRM concentration of nuclear area non-specific binding; for the free TMRM concentration in nuclear area, its value and the born of the same parents TMRM concentration of dissociating outward equates; Then, the free TMRM concentration in the TMRM concentration of the nuclear area non-specific binding calculating and nuclear area is utilized Matlab pass through equation (3) the model of two independent binding sites of an aglucon carry out matching:

$\left[{\mathrm{TMRM}}_B^{+}\right]=\frac{\left[{\mathrm{TMRM}}_{B\max 1}^{+}\right]\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}{K_{D1}+\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}+\frac{\left[{\mathrm{TMRM}}_{B\max 2}^{+}\right]\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}{K_{D2}+\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}---\left(3\right)$

[TMRM wherein ⁺ _bmax1] and [TMRM ⁺ _bmax2] be respectively the TMRM concentration of maximum non-specific binding in two independent binding sites, K _d1and K _d2be respectively the dissociation constant of TMRM and two independent binding sites, try to achieve [TMRM ⁺ _bmax1]=22780nM, [TMRM ⁺ _bmax2]=5348nM, K _d1=38140nM, K _d2=988.2nM, R ²=0.9949, illustrate that fitting effect is fine.

The Scatchard figure that in Fig. 2, a obtains after matching for utilizing equation (2) to carry out.Figure Zhong “ ﹍ " with “ ﹎ " dotted line represents respectively two independent binding sites in equation, the effect of carrying out matching with single independent binding site is as seen poor.Illustration is the TMRM fluorescence intensity of non-specific adsorption and the graph of a relation of free TMRM, and at free TMRM area with high mercury, the TMRM fluorescent value of non-specific adsorption exists obvious saturated phenomenon as seen.From the above results, with two independent binding sites, at high concentration and the low concentration region of TMRM, can well to the TMRM of non-specific adsorption, demarcate.With certain density TMRM solution incubated cell, measure the TMRM fluorescence intensity of nuclear area, through type (1) calculates nuclear area TMRM total concentration, and equation (3) substitution equation (2) is solved and obtained can utilize Nernst equation (4) to calculate cell resting membrane electric potential:

$V_m=-60\mathrm{lo}{g}_{10}\frac{\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}{\left[{\mathrm{TMRM}}_o^{+}\right]}---\left(4\right)$

V wherein _mfor RMP, [TMRM _o ⁺] be extracellular TMRM concentration.

RMP detection method of the present invention is to set up an average non-specific adsorption level of formerly demarcating cell to be measured, then carries out RMP detection with it.Because the non-specific adsorption between cell individual has different, therefore, concerning individual cells, may there is certain error in its value, but for a cell colony, its accuracy should be higher, hereinafter also will verify its accuracy.For different cells, its non-specific adsorption is also possible, and there is some difference, but should demarcate comparatively accurately with said method.It is to be noted simultaneously, in free TMRM low strength range (for C17.2 cell, be less than 1500nM), by the fixedly non-specific adsorption ratio of bibliographical information, carry out matching, also there is higher precision, but this is confined to detect the cell of some RMP calibrations, in nuclear area, free TMRM relative concentration is lower.

2, RMP detects and patch-clamp checking during gradient depolarization

To verify to the accuracy of RMP detection method of the present invention first-selected patch-clamp method.Yet for the cell of adherent growth in normal solution, generally need suitably to digest the sealing-in that is beneficial to electrode, especially for this adherent thinner cell of C17.2; Simultaneously, sealing-in will cause certain damage to cell, add liquid in top electrode and will change the original ion concentration of intracellular fluid, there is certain difference in the RMP value that therefore patch-clamp detects and the RMP value of actual cell, and detect by TMRM method, there are not the problems referred to above, therefore cannot accurately contrast with the data that patch-clamp detects.In order to address this problem, the present invention detects one group of unpolarized cell RMP of gradient, with the gradient K that adds valinomycins ⁺hBS solution (K ⁺concentration is respectively 5,7.5,15,35,55,80,130mM, gradient K herein ⁺hBS solution, except special instruction, is the gradient of above-mentioned numerical value; Gradient K herein ⁺in HBS solution, with Choline Chloride, substitute NaCl, Choline Chloride and potassium concentration summation are 135mM) depolarization cell, because born of the same parents do not have sodion outward, and valinomycins also has very little permeability to sodion, add row's sodium behavior of cell itself, therefore in cell, sodion can run off gradually, and the summation that in born of the same parents, potassium concentration can approach born of the same parents' outer cationic concentration is 135mM (in born of the same parents, outer cationic summation is substantially equal), now by becoming in born of the same parents, potassium ion approaches 135mM to cell and potassium ion is this special cell distributing of complete energy.

Detection method of the present invention is as follows: C17.2 cell is cultivated 3 days on 25mm circular lid slide, put in stainless steel double dish, with the 5mM K that adds 1 μ M valinomycins ⁺hBS solution (substituting NaCl with Choline Chloride) is hatched after 1 hour in 37 ℃, puts on 37 ℃ of warm tables of laser confocal microscope, and the visual field immobilizes after being adjusted to the position with suitable cell density, respectively with the gradient K that contains 500nM TMRM ⁺hBS solution (substituting NaCl with Choline Chloride) is hatched 25min, then the same visual field is taken, obtain apart from the fluorogram at substrate of glass 3 μ m places, utilize AXIOVISION software to carry out the analysis of fluorescence gray-scale value to the same nuclear area of every figure, and then obtain corresponding RMP by equation (4).

Patch-clamp verification method is as follows: C17.2 cell is cultivated 3 days in 35mm double dish, with the 5mM K that adds 1 μ M valinomycins ⁺hBS solution (substituting NaCl with Choline Chloride) is hatched after 1 hour in 37 ℃, uses respectively gradient K ⁺hBS solution (substituting NaCl with Choline Chloride) rinses double dish dipping bath cell.With the method for Perforated patch clamp, eletrode tip sucks 30 μ g/mL amphotericin Bs in advance, then pours into the interior liquid of electrode (by 135mM KCl, 20mM HEPES, 10mM EGTA, 1mM CaCl ₂with 2mM Na ₂aTP forms), find under the microscope the good cell of form, the complete above sealing-in in cell pattern 1G Europe, records RMP by EPC-10 patch clamp amplifier (German HEKA) with current clamp.

In Fig. 2, b adopts respectively the inventive method and patch-clamp method to detect the result of C17.2 cell RMP after gradient depolarization.From scheming, the detected value of the inventive method and the detected value of patch-clamp and theoretical value are basic identical, and wherein the calculating of theoretical value is that 135mM and potassium ion are this special distribution of complete energy based on potassium ion in born of the same parents.The above results explanation RMP detection method based on TMRM of the present invention has good accuracy.

Four, P _na/Kdetection

1, in born of the same parents, Na ion concentration detects during gradient depolarization

Owing to cannot change the ion concentration of intracellular fluid as patch-clamp, therefore calculating P _na/Kfront needs first detect sodium, potassium concentration in born of the same parents.The present invention is based on sodion fluorescent dye CoroNa Green Na ion concentration in born of the same parents is detected, then by Na ion concentration, extrapolate potassium concentration.

Concrete grammar is as follows: C17.2 cell is cultivated 3 days on 25mm circular lid slide, put in stainless steel double dish, with the 5mM K that adds 1 μ M valinomycins, 10 μ M cobans and 5 μ M CoroNa Green (Invitrogen, the U.S.) ⁺hBS solution is hatched after 1 hour in 37 ℃, put on 37 ℃ of warm tables of laser confocal microscope, the visual field immobilizes after being adjusted to the position with suitable cell density, respectively with containing 10mM, 25mM, 55mM, 80mM, 100mM, 120mM, the HBS solution of 130mM sodion is hatched 25min, then adopt the argon laser of 488nm and the band of 505-550nm to lead to filter disc (peak excitation wavelength/emission wavelength of CoroNa Green is 492nm/516nm) the same visual field is carried out to laser confocal imaging, obtain apart from the fluorogram at substrate of glass 3 μ m places, utilize AXIOVISION software to carry out the analysis of green fluorescence gray-scale value to cell centre region, draw CoroNa Green calibration curve, by Matlab, following formula is carried out to matching:

$\left[{\mathrm{Na}}_i^{+}\right]=\frac{K_d(F-F_{\min })}{F_{\max }-F}---\left(5\right)$

[Na wherein _i ⁺] be Na ion concentration in the born of the same parents under corresponding fluorescence intensity, K _dfor the dissociation constant of sodion and CoroNa Green, F is the fluorescence intensity level under corresponding Na ion concentration, F _minfor not containing the fluorescence intensity level (obtaining by not containing the HBS solution of sodion) of sodion, F _maxfor the fluorescence intensity level containing saturated sodion (by adding the HBS solution containing 1000mM sodion to make to obtain under the complete saturated situation of CoroNa Green in cell).

In Fig. 3, a is CoroNa Green calibration curve (n=24), and illustration is CoroNa Green Color figure.By Matlab, formula (5) is carried out to matching, try to achieve the dissociation constant K of sodion and CoroNa Green _dvalue is 86.98 ± 10.19mM.

Then detect as stated above the cultivation C17.2 cell of 3 days at the gradient K that only adds 5 μ M CoroNa Green ⁺in HBS solution fluorescence intensity, through type (5) calculates Na ion concentration in born of the same parents, recycling Matlab carries out matching to following formula:

$\left[{\mathrm{Na}}_i^{+}\right]=\mathrm{\&Phi;}\left(\right[K_o^{+}\left]\right)---\left(6\right)$

[K wherein _o ⁺] be the outer potassium concentrations of born of the same parents.

The graph of a relation of the interior Na ion concentration of born of the same parents and the outer potassium concentration of born of the same parents when b is gradient depolarization in Fig. 3.From scheming, along with the outer potassium concentration of born of the same parents increases, namely born of the same parents' extracellular sodium ion concentration reduces, in born of the same parents, Na ion concentration also decreases, estimation is to cause because infiltration rate degree in sodion slows down, so in born of the same parents, potassium concentration also can correspondingly raise, to maintain the normal osmotic pressure balance of cell.

The empirical equation that obtains Na ion concentration in the outer potassium concentration of born of the same parents and born of the same parents is as follows:

$\left[{\mathrm{Na}}_i^{+}\right]=-0.2287\left[K_o^{+}\right]+38.701.$

Above-mentioned equation (6) is to be based upon on the basis of asking the somatic average of group, and uses it for the hereinafter calculating of sodium potassium ion permeability ratio.This is because CoroNa Green dyestuff has a small amount of red fluorescence and is excited, thereby affect the dye accuracy of data of TMRM, and vice versa, so CoroNa Green and the TMRM cell that can not simultaneously dye, and can only detect in advance its [Na _i ⁺].

2, P under the outer high potassium of gradient born of the same parents _na/Kcalculate

RMP can be calculated by Goldman equation.Because chlorion is less to the contribution of RMP, Goldman equation can be reduced to:

$V_m=-60\mathrm{lo}{g}_{10}\frac{\left[K_i^{+}\right]+P_{\mathrm{Na}/K}\left[{\mathrm{Na}}_i^{+}\right]}{\left[K_o^{+}\right]+P_{\mathrm{Na}/K}\left[{\mathrm{Na}}_o^{+}\right]}---\left(7\right)$

[Na wherein _i ⁺], [K _i ⁺] be respectively sodium in born of the same parents, potassium concentration, [Na _o ⁺], [K _o ⁺] be respectively the outer sodium of born of the same parents, potassium concentration.

By equation (6) substitution equation (7), think that the inside and outside kalium ion and natrium ion concentration summation of born of the same parents equates, therefore [K simultaneously _i ⁺]=135-[Na _i ⁺], V _mcan be by [K _o ⁺] represent to set up following equation:

$V_m=-60{\log }_{10}\frac{135-\mathrm{\&Phi;}\left(\right[K_o^{+}\left]\right)+P_{\mathrm{Na}/K}\mathrm{\&Phi;}\left(\right[K_o^{+}\left]\right)}{\left[K_o^{+}\right]+P_{\mathrm{Na}/K}(135-[K_o^{+}\left]\right)}---\left(8\right)$

Therefore, calculating in born of the same parents on the basis of Na ion concentration, with the gradient concentration potassium ion solution incubated cell that contains finite concentration TMRM, measure the TMRM fluorescence intensity of nuclear area, by equation (4), calculate corresponding RMP, then by RMP and born of the same parents outer for potassium concentration equation (8) carry out matching, can obtain P _na/K.Concrete grammar is as follows: C17.2 cell is cultivated 3 days on 25mm circular lid slide, put in stainless steel double dish, and be put on 37 ℃ of warm tables of laser confocal microscope, first clap and get background fluorescence picture, then add respectively the gradient K that contains 500nM TMRM ⁺hBS solution is hatched 25min, the same visual field is taken, obtain apart from the fluorogram at substrate of glass 3 μ m places, utilize AXIOVISION software to carry out the analysis of fluorescence gray-scale value to the same nuclear area of every figure, by equation (4), obtain corresponding RMP, then by Matlab, equation (8) is carried out to matching, obtain the P of colony _na/Kvalue.Meanwhile, calculate 7 kinds of outer K not born of the same parents ⁺independent P during concentration _na/Kvalue, with the P of colony _na/Kvalue compares analysis.

During depolarization, RMP data, as shown in c in Fig. 3, utilize Matlab to obtain the P of colony to its matching _na/Kvalue is 0.015 ± 0.004.The degree that departs from potassium ion Nernst curve for the ease of understanding cell, has dotted K in born of the same parents in c in Fig. 3 ⁺k in Nernst curve when concentration is fixed as 135mM and born of the same parents ⁺concentration is passed through 135-[Na _i ⁺] K in the born of the same parents that calculate ⁺the Nernst curve of concentration change.

Corresponding 4 kinds of outer K not born of the same parents ⁺concentration (K ⁺concentration is respectively 5,7.5,15,35mM) independent P _na/Kvalue is as shown in d in Fig. 3, due to the outer K of born of the same parents ⁺concentration is the P of 55～130mM _na/Kthe standard deviation of value is excessive, and data have lost meaning, therefore show in the drawings.From scheming, 4 groups of K of 5～35mM ⁺under concentration gradient, P _na/Kthe standard deviation of value is amplified fast, and this is P _na/Kthe Systematic Errors that computing method cause, because P in the present invention _na/Kvalue is obtained by RMP, and identical RMP standard deviation is being converted into P _na/Kafter, the RMP of calibration will produce larger P _na/Kstandard deviation, the P while calculating High K+ depolarization by patch-clamp method _na/Ktime also there is same problem, therefore from these data, cannot determine P _na/Kwhether with potassium concentration, increase that (variance analysis is without significant difference, p>0.05).The outer K of more single born of the same parents but ⁺p under concentration _na/Kthe P of colony that value and overall fit obtain _na/Kvalue is found, 5mM K ⁺under P _na/Kvalue is basic consistent with overall fit value, visible 5mM K ⁺under P _na/Kvalue detects the P that can represent comparatively accurately cell _na/Klevel.

In neural stem cell, this two class of wide expression K2P, Kir continues or the potassium-channel of long period in state of activation at quiescent condition, and this has determined the permeability size of potassium ion on cell membrane.Yet do not have bibliographical information in neural stem cell, to have the sodium channel of sustained activation, for example Nalcn.Hence one can see that, and the sodium in neural stem cell on cell membrane and potassium permeability are all provided by the potassium channel of sustained activation.Because above-mentioned two class potassium channels are very high to the selectivity of potassium ion, cause P on cell _na/Kvery low, the value of bibliographical information is less than 0.04, the P that this and the present invention calculate _na/Kbe worth basically identical.

Five, the RMP based on TMRM and P _na/Kthe application detecting

1, RMP and the P in the different propagation of cell period _na/Kdetect

C17.2 cell is cultivated respectively 1,2,3 day on 25mm circular lid slide, put in stainless steel double dish, and be put on 37 ℃ of warm tables of laser confocal microscope, first clap and get background fluorescence picture, then add the 5mMK containing 500nM TMRM ⁺hBS solution is hatched 25min, and the same visual field is taken, and obtains apart from the fluorogram at substrate of glass 3 μ m places, utilizes AXIOVISION software to carry out the analysis of fluorescence gray-scale value, obtains respectively the cell RMP cultivating 1,2,3 day by equation (4).The cell in above-mentioned different propagation period is carried out to RMP record with Perforated patch clamp method, in electrode, liquid is by 130mM KCl, 5mM NaCl, 20mM HEPES, 10mM EGTA, 1mM CaCl simultaneously ₂with 2mM Na ₂aTP forms.While recycling aforementioned gradient depolarization, Na ion concentration detection method is carried out Na ion concentration in born of the same parents to the cell in above-mentioned different propagation period and is detected in born of the same parents, in conjunction with equation (8), calculates C17.2 cell at the difference propagation P in period _na/K.

The RMP that in Fig. 4, a is the different propagation of cell period of adopting respectively the inventive method and recording from patch-clamp method, two of each cultivated days kinds of methods are recorded to numerical value and carry out Student ' st check discovery all without significant difference (p>0.05), and two kinds of all reductions (p<0.01) significantly day by day of RMP value that method records under different cultivated days.In Fig. 4, b is for cultivating 1, 2, Na ion concentration in the born of the same parents of the C17.2 cell of 3 days, be presented at the interior Na ion concentration of Initial stage of culture born of the same parents higher, and along with the increase of incubation time, in born of the same parents, Na ion concentration reduces (p<0.05) significantly day by day, when its possible cause is passage, the processing procedures such as trypsinization reduce the metaboilic level of cell, and then affect row's sodium efficiency of NaK pump, Na ion concentration in born of the same parents is raise, and along with cell again adherent growth, its metaboilic level recovers gradually, NaK pump also recovers normal work efficiency, thereby Na ion concentration in born of the same parents is reduced gradually.In born of the same parents, higher Na ion concentration shows that in born of the same parents, potassium concentration is accordingly in reduced levels, and this is also one of reason causing as shown in a in Fig. 4 Initial stage of culture RMP calibration.The P of c for adopting the inventive method and patch-clamp method to record respectively in Fig. 4 _na/Kvalue, finds two kinds of P that method records under different cultivated days _na/Kvalue all reduces (p<0.05) significantly day by day, and two kinds of methods data when cultivating 1,2 day are without significant difference (p>0.05), and the P that TMRM method detects while cultivating 3 days _na/Kvalue is significantly less than patch-clamp detected value (p<0.001), this may be because patch-clamp while detecting for making cell rounding be beneficial to electrode sealing-in, need to carry out suitable trypsinization to cell, the cause that may cause cell membrane upper channel albumen to a certain degree to damage, and when cultivating 1,2 day due to P _na/Kvalue demonstrates more greatly and not this problem.Above-mentioned experimental result explanation detection method of the present invention can be carried out RMP and sodium potassium ion permeability ratio comparatively accurately to plane cell and be detected, and compares patch-clamp method and have higher detection flux, can to a large amount of cells, detect fast.

2, RMP and the P of the cell of three-dimensional aggregation growth _na/Kdetect

It is 80 μ m that the present invention has designed small recessed diameter, and channel width is respectively the small recessed graphic of 40 μ m, and the degree of depth is 100 μ m.Graphic basic structure is to consist of the small recessed unit of channel attached 5-linked repeated arrangement, and every kind of structure macro-size is the square of length of side 1cm.Utilize traditional soft lithographic processing technology to make PDMS (Sylgard 184, Dow Corning, the U.S.) small recessed graphic.Preparation process is as follows: silicon chip (P type <100>, resistivity 7-13 Ω/cm ², Beijing Grinm Semiconductor material Co., Ltd) surface first by oxidation, photoresist coating, ultraviolet light photoetching development, hydrofluorite wet etching, the etching of BOSCH technique dry method silicon, remove photoresist coating and be processed into the main mould of silicon; Then use 1H, 1H, 2H, 2H-perfluoro capryl trichlorosilane (Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane 97%, Sigma-Aldrich, the U.S.) release agent is processed 30 minutes in a vacuum, makes the main mould surface silicon of silicon alkanisation; By PDMS performed polymer and hardening agent in mass ratio 10:1 mix, after vacuum stripping, be poured on the main mould of silicon of surface silicon alkanisation, again after vacuum stripping in 50 ℃ of heating 3 hours, peel off the main mould of silicon after cooling, obtaining PDMS, to connect hole small recessed graphic.PDMS planar structure directly makes by pouring into PDMS on smooth silicon chip.

By density, be 1 * 10 ⁵the C17.2 cell of/mL is inoculated in containing in the small recessed graphic confocal special-purpose capsule of PDMS, with amplification culture medium, cultivate after 3 days, to the three-dimensional cell aggregation in small recessed graphic middle formation, in born of the same parents, Na ion concentration detection method is carried out Na ion concentration in born of the same parents and is detected during with aforementioned gradient depolarization, use again the method based on TMRM to record cell RMP, by equation (8), calculate P _na/K, contrast with PDMS planar structure cultured cell simultaneously.

Result as shown in Figure 5, finds that its RMP value is significantly less than plane cultured cells (p<0.001) at the cell of the three dimensional growth of small recessed middle cultivation, and P _na/Ksignificantly be greater than plane cultured cells (p<0.001), this explanation three dimensional growth its RMP of cell than the cell of two-dimensional growth want polarization some.Other scholars also studies have found that cellular electrophysiologicalsensor characteristic can there are differences under three peacekeeping two-dimensional growth states, prompting may be that the difference of the growth dimension of cell causes the difference that sodium potassium-channel is expressed, different Growth of Cells dimensions also affects the contact area of cell and liquid simultaneously, and then affects ion by the efficiency of ion channel.

Because cell is three dimensional stress suspension growth small in recessed, with the method for patch-clamp is more difficult, cell under this growth conditions is carried out to sealing-in, to the cell of aggregation inside, can not detect especially.And the method based on TMRM that the present invention sets up can well detect the cell of small recessed middle three dimensional growth.

Six, the inventive method and the existing methodical contrast based on TMRM

1, contrast traditional RMP detection method based on TMRM

In order to understand more comprehensively the inventive method, the RMP detection method based on TMRM of the foundation such as itself and Leow is compared.

The ratio of the theory of Leow method based on represent in born of the same parents TMRM total concentration in free TMRM concentration and born of the same parents with a fixed coefficient f, and then the non-specific adsorption in demarcation core district; With the 130mM K that adds valinomycins ⁺hBS solution makes cell depolarization arrive zero potential (V _m=0), due to TMRM concentration and its fluorescence intensity linear, so can calculate f value with following equation:

$V_m=-60\mathrm{lo}{g}_{10}\frac{f\left[F_i\right]}{\left[F_o\right]}---\left(9\right)$

[F wherein _i], [F _o] be respectively the inside and outside TMRM fluorescence intensity of born of the same parents.After acquisition f value, be the RMP that available formula (9) is calculated cell under different conditions.

RMP while recalculating aforementioned cell gradient depolarization by above-mentioned Leow method, RMP and the P in the different propagation of cell period _na/K, and RMP and the P of the cell of three-dimensional aggregation growth _na/K, be then analyzed with the calculated value of the inventive method.Result is as follows: by the 130mM K to interpolation valinomycins ⁺the fluorescence data analysis of HBS solution group, obtaining non-specific adsorption factor f value is 0.156 ± 0.047; With this to 5～130mM K of hatching with valinomycins ⁺hBS solution gradient depolarization data are verified, result is as shown in square marks in a in Fig. 6, obviously along with the change of current potential is negative, it is also increasing that it departs from standard Nernst curve, this is because when current potential is negative, nuclear area non-specific adsorption is tending towards saturated, thereby non-specific adsorption factor f value will raise gradually, and fixing f value will be used the RMP value polarization of its calculating; At the 5～130mM K to not hatching with valinomycins ⁺hBS solution gradient depolarization data are calculated and are also obtained similar results (b in Fig. 6), and and then cause the 5～35mM calculating respectively to organize P _na/Kcompare the calculated value (p<0.001) significantly bigger than normal (c in Fig. 6) of the inventive method.Because Leow method is comparatively convenient when expressing non-specific adsorption, therefore when the cell detecting compared with positive potential, can consider this method.

2, the method that contrast inventor place seminar sets up in earlier stage

Inventor place seminar has set up a kind of P based on TMRM early stage on the basis of Leow method _na/Kdetection method (Chinese patent ZL201110218448.7), the method is with the 5～130mM K that adds valinomycins ⁺hBS solution (replacing NaCl with Choline Chloride) gradient depolarization cell is supposed potassium concentration [K in born of the same parents simultaneously _i ⁺] constant and unknown, because potassium ion inside and outside cell under valinomycins effect is in this special balanced distribution of complete energy, can obtain:

$\frac{f\left[F_i\right]}{\left[F_o\right]}=\frac{\left[K_i^{+}\right]}{\left[K_o^{+}\right]}---\left(10\right)$

To 5～130mM K ⁺6 groups of data with above-mentioned equation, carry out Matlab matching and obtain a fixing [K _i ⁺]/f value.While using with similar approach of the present invention depolarization, in born of the same parents, Na ion concentration detects simultaneously, obtains a mean value.Then use 5～130mM K ⁺hBS solution carries out gradient depolarization to cell, obtains following equation on the basis of formula (7):

$\frac{\left[F_i\right]}{\left[F_o\right]}=\frac{\left[K_i^{+}\right]/f+P_{\mathrm{Na}/K}\left[{\mathrm{Na}}_i^{+}\right]/f}{\left[K_o^{+}\right]+P_{\mathrm{Na}/K}\left[{\mathrm{Na}}_o^{+}\right]}---\left(11\right)$

By acquired [K _i ⁺]/f value substitution equation (11), obtains f value and P with Matlab matching _na/Kvalue.And then can calculate [K _i ⁺] value and the RMP value of cell during gradient depolarization.

RMP while recalculating aforementioned cell gradient depolarization with said method, and RMP and the P in the different propagation of cell period _na/K, be then analyzed with the calculated value of the inventive method.Result is as follows: with the 5～130mM K that adds valinomycins ⁺hBS solution (replacing NaCl with Choline Chloride) gradient depolarization cell the data obtained carries out matching to formula (10), obtains [K _i ⁺]/f value is 431.6mM.Na ion concentration in born of the same parents is detected and found, at 5～35mM K ⁺time born of the same parents in Na ion concentration comparatively stable, mean value is 35.10mM; By above-mentioned 2 result substitution equations (11), and to 5～35mM K ⁺gradient data carries out matching, obtains P _na/K=0.078, f=0.090; By this f value to 5～35mM K ⁺gradient data carries out RMP calculating, obtains data as shown in circular mark in b in a, Fig. 6 in Fig. 6, finds all calculated values that departs from the inventive method and Leow method by a relatively large margin of its RMP value; With f value substitution [K _i ⁺]/f value calculates [K _i ⁺]=38.77mM, value when this is worth far below the neural stem cell normal growth of bibliographical information, shows that the method may exist some problems.To 5～35mM K ⁺gradient data carries out P _na/Kcalculate, result as shown in c in Fig. 6,5～15mM K wherein ⁺group is compared (p<0.001) significantly bigger than normal with the calculated value of the inventive method.The reasons for the above problems have 2 points: (1) supposes [K in born of the same parents _i ⁺] and [Na _i ⁺] constant, (2) carry out matching with a fixing f value.

Finally explanation is, above preferred embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is described in detail by above preferred embodiment, but those skilled in the art are to be understood that, can to it, make various changes in the form and details, and not depart from the claims in the present invention book limited range.

Claims (10)

1. the cell resting membrane electric potential detection method based on TMRM, is characterized in that, comprises the following steps:

The relation of A.TMRM fluorescence intensity and concentration is demarcated

TMRM is mixed with to the solution of a series of gradient concentrations, above-mentioned solution is carried out respectively to fluorescence intensity detection, by the TMRM fluorescence intensity recording and corresponding TMRM for concentration formula (1) carry out matching:

F＝k[TMRM ⁺] (1)

Wherein F is TMRM fluorescence intensity, [TMRM ⁺] be TMRM concentration; Try to achieve calibration coefficient k;

B. the non-specific adsorption of nuclear area TMRM is demarcated

First with potassium, penetrating dose of processing cell of sodion, make the complete depolarization of cell, the inside and outside free TMRM concentration of cell equates, with the TMRM solution of a series of gradient concentrations, distinguish incubated cell again, measure nuclear area TMRM fluorescence intensity, through type (1) calculates nuclear area TMRM total concentration, then through type (2) calculates the TMRM concentration of nuclear area non-specific binding:

$\left[{\mathrm{TMRM}}_i^{+}\right]=\left[{\mathrm{TMRM}}_B^{+}\right]+\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]---\left(2\right)$

[TMRM wherein _i ⁺] be nuclear area TMRM total concentration, [TMRM _b ⁺] be the TMRM concentration of nuclear area non-specific binding; for the free TMRM concentration in nuclear area, its value and the born of the same parents TMRM concentration of dissociating outward equates;

Then, by the free TMRM concentration of the TMRM concentration of the nuclear area non-specific binding calculating and nuclear area, with the model of two independent binding sites of an aglucon, be that formula (3) is carried out matching:

$\left[{\mathrm{TMRM}}_B^{+}\right]=\frac{\left[{\mathrm{TMRM}}_{B\max 1}^{+}\right]\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}{K_{D1}+\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}+\frac{\left[{\mathrm{TMRM}}_{B\max 2}^{+}\right]\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}{K_{D2}+\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}---\left(3\right)$

[TMRM wherein ⁺ _bmax1] and [TMRM ⁺ _bmax2] be respectively the TMRM concentration of maximum non-specific binding in two independent binding sites, K _d1and K _d2be respectively the dissociation constant of TMRM and two independent binding sites; Try to achieve [TMRM ⁺ _bmax1], [TMRM ⁺ _bmax2], K _d1and K _d2;

C. the cell resting membrane electric potential based on TMRM detects

With certain density TMRM solution incubated cell, measure the TMRM fluorescence intensity of nuclear area, through type (1) calculates nuclear area TMRM total concentration, and formula (3) substitution formula (2) is solved and obtained through type (4) calculates cell resting membrane electric potential again:

$V_m=-60\mathrm{lo}{g}_{10}\frac{\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}{\left[{\mathrm{TMRM}}_o^{+}\right]}---\left(4\right)$

V wherein _mfor cell resting membrane electric potential, [TMRM _o ⁺] be extracellular TMRM concentration.

2. the cell resting membrane electric potential detection method based on TMRM as claimed in claim 1, is characterized in that, is to contain 5mM KCl, 130mM NaCl, 1mM MgCl in steps A ₂, 2mM CaCl ₂with the 20mM HEPES damping fluid of 10mM glucose be solvent, TMRM is mixed with to concentration is respectively 5,50,100,250,500,1500,2500,3500,5000,10000, the solution of 50000nM.

3. the cell resting membrane electric potential detection method based on TMRM as claimed in claim 1, it is characterized in that, penetrating dose of potassium described in step B, sodion are respectively valinomycins and coban, first with containing 1 μ M valinomycins, 10 μ M cobans, 135mM KCl, 1mM MgCl ₂, 2mM CaCl ₂process cell with the 20mM HEPES damping fluid of 10mM glucose and make the complete depolarization of cell, then to contain 135mM KCl, 1mM MgCl ₂, 2mM CaCl ₂be respectively 500,1500,5000,10000 with the 20mMHEPES damping fluid of the 10mM glucose concentration that is solvent preparation, the TMRM solution incubated cell of 20000nM.

4. the cell resting membrane electric potential detection method based on TMRM as claimed in claim 1, is characterized in that, described in step C, with certain density TMRM solution incubated cell, is to contain 5mM KCl, 130mM NaCl, 1mM MgCl ₂, 2mM CaCl ₂with the 20mM HEPES damping fluid of the 10mM glucose concentration that is solvent preparation the TMRM solution incubated cell that is 500nM.

5. the sodium potassium ion permeability based on TMRM, than value detection method, is characterized in that, comprises the following steps:

The relation of A.TMRM fluorescence intensity and concentration is demarcated

TMRM is mixed with to the solution of a series of gradient concentrations, above-mentioned solution is carried out respectively to fluorescence intensity detection, by the TMRM fluorescence intensity recording and TMRM for concentration formula (1) carry out matching:

F＝k[TMRM ⁺] (1)

Wherein F is TMRM fluorescence intensity, [TMRM ⁺] be TMRM concentration; Try to achieve calibration coefficient k;

B. the non-specific adsorption of nuclear area TMRM is demarcated

First with potassium, penetrating dose of processing cell of sodion, make the complete depolarization of cell, the inside and outside free TMRM concentration of cell equates, with the TMRM solution of a series of gradient concentrations, distinguish incubated cell again, measure nuclear area TMRM fluorescence intensity, through type (1) calculates nuclear area TMRM total concentration, then through type (2) calculates the TMRM concentration of nuclear area non-specific binding:

$\left[{\mathrm{TMRM}}_i^{+}\right]=\left[{\mathrm{TMRM}}_B^{+}\right]+\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]---\left(2\right)$

[TMRM wherein _i ⁺] be nuclear area TMRM total concentration, [TMRM _b ⁺] be the TMRM concentration of nuclear area non-specific binding; for the free TMRM concentration in nuclear area, its value and the born of the same parents TMRM concentration of dissociating outward equates;

Then, by the free TMRM concentration of the TMRM concentration of the nuclear area non-specific binding calculating and nuclear area, with the model of two independent binding sites of an aglucon, be that formula (3) is carried out matching:

$\left[{\mathrm{TMRM}}_B^{+}\right]=\frac{\left[{\mathrm{TMRM}}_{B\max 1}^{+}\right]\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}{K_{D1}+\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}+\frac{\left[{\mathrm{TMRM}}_{B\max 2}^{+}\right]\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}{K_{D2}+\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}---\left(3\right)$

[TMRM wherein ⁺ _bmax1] and [TMRM ⁺ _bmax2] be respectively the TMRM concentration of maximum non-specific binding in two independent binding sites, K _d1and K _d2be respectively the dissociation constant of TMRM and two independent binding sites; Try to achieve [TMRM ⁺ _bmax1], [TMRM ⁺ _bmax2], K _d1and K _d2;

C. the cell resting membrane electric potential based on TMRM detects

With certain concentration TMRM solution incubated cell, measure the TMRM fluorescence intensity of nuclear area, through type (1) calculates nuclear area TMRM total concentration, and formula (3) substitution formula (2) is solved and obtained through type (4) calculates cell resting membrane electric potential again:

$V_m=-60\mathrm{lo}{g}_{10}\frac{\left[{\mathrm{TMRM}}_{\mathrm{if}}^{+}\right]}{\left[{\mathrm{TMRM}}_o^{+}\right]}---\left(4\right)$

V wherein _mfor cell resting membrane electric potential, [TMRM _o ⁺] be extracellular TMRM concentration;

D. in born of the same parents, Na ion concentration detects during gradient depolarization

First with potassium, penetrating dose of processing cell of sodion, make the inside and outside sodion balance of cell, cell is loaded to sodion fluorescent dye CoroNa Green simultaneously, with the sodion solution of a series of gradient concentrations, distinguish incubated cell again, measure cell fluorescence intensity, by fluorescence intensity and born of the same parents for Na ion concentration formula (5) carry out matching:

$\left[{\mathrm{Na}}_i^{+}\right]=\frac{K_d(F-F_{\min })}{F_{\max }-F}---\left(5\right)$

[Na wherein _i ⁺] be Na ion concentration in the born of the same parents under corresponding fluorescence intensity, F is the fluorescence intensity level under corresponding Na ion concentration; F _minfor not containing the fluorescence intensity level of sodion, by obtaining with the solution incubated cell that does not contain sodion; F _maxfor containing the fluorescence intensity level of saturated sodion, in cell, in the complete saturated situation of CoroNa Green, obtain by making with 1000mM sodion solution incubated cell; Try to achieve the dissociation constant K of sodion and CoroNa Green _d;

Then, detect as stated above the fluorescence intensity of cell in the gradient concentration potassium ion solution that adds CoroNa Green do not use potassium, penetrating dose of processing of sodion, the fluorescence intensity substitution formula (5) recording is calculated to Na ion concentration in born of the same parents, then by the outer potassium concentration of born of the same parents and born of the same parents for Na ion concentration formula (6) carry out matching:

$\left[{\mathrm{Na}}_i^{+}\right]=\mathrm{\&Phi;}\left(\right[K_o^{+}\left]\right)---\left(6\right)$

[K wherein _o ⁺] be the outer potassium concentrations of born of the same parents; Try to achieve Φ;

E. sodium potassium ion permeability ratio detects

Cell resting membrane electric potential can calculate with the Goldman equation of simplifying:

$V_m=-60\mathrm{lo}{g}_{10}\frac{\left[K_i^{+}\right]+P_{\mathrm{Na}/K}\left[{\mathrm{Na}}_i^{+}\right]}{\left[K_o^{+}\right]+P_{\mathrm{Na}/K}\left[{\mathrm{Na}}_o^{+}\right]}---\left(7\right)$

[K wherein _i ⁺] be potassium concentration in born of the same parents, [Na _o ⁺] be born of the same parents' extracellular sodium ion concentration; Corresponding different [K _o ⁺] [Na _i ⁺] can be obtained by formula (6); The in the situation that inside and outside cell, sodium potassium concentration summation being equal, V _mcan be by [K _o ⁺] represent:

$V_m=-60{\log }_{10}\frac{135-\mathrm{\&Phi;}\left(\right[K_o^{+}\left]\right)+P_{\mathrm{Na}/K}\mathrm{\&Phi;}\left(\right[K_o^{+}\left]\right)}{\left[K_o^{+}\right]+P_{\mathrm{Na}/K}(135-[K_o^{+}\left]\right)}---\left(8\right)$

Wherein, P _na/Kfor sodium potassium ion permeability ratio;

With the gradient concentration potassium ion solution incubated cell that contains finite concentration TMRM, measure the TMRM fluorescence intensity of nuclear area, through type (4) calculates corresponding cell resting membrane electric potential, then by cell resting membrane electric potential and born of the same parents outer for potassium concentration formula (8) carry out matching, can obtain P _na/K.

6. the sodium potassium ion permeability based on TMRM as claimed in claim 5, than value detection method, is characterized in that, is to contain 5mM KCl, 130mM NaCl, 1mM MgCl in steps A ₂, 2mM CaCl ₂with the 20mMHEPES damping fluid of 10mM glucose be solvent, TMRM is mixed with to concentration is respectively 5,50,100,250,500,1500,2500,3500,5000,10000, the solution of 50000nM.

7. the sodium potassium ion permeability based on TMRM as claimed in claim 5 compares value detection method, it is characterized in that, penetrating dose of potassium described in step B, sodion are respectively valinomycins and coban, first with containing 1 μ M valinomycins, 10 μ M cobans, 135mM KCl, 1mM MgCl ₂, 2mM CaCl ₂process cell with the 20mM HEPES damping fluid of 10mM glucose and make the complete depolarization of cell, then to contain 135mM KCl, 1mM MgCl ₂, 2mM CaCl ₂be respectively 500,1500,5000,10000 with the 20mM HEPES damping fluid of the 10mM glucose concentration that is solvent preparation, the TMRM solution incubated cell of 20000nM.

8. the sodium potassium ion permeability based on TMRM as claimed in claim 5, than value detection method, is characterized in that, described in step C, with certain density TMRM solution incubated cell, is to contain 5mM KCl, 130mM NaCl, 1mM MgCl ₂, 2mM CaCl ₂with the 20mM HEPES damping fluid of the 10mM glucose concentration that is solvent preparation the TMRM solution incubated cell that is 500nM.

9. the sodium potassium ion permeability based on TMRM as claimed in claim 5 compares value detection method, it is characterized in that, penetrating dose of potassium described in step D, sodion are respectively valinomycins and coban, first with containing 1 μ M valinomycins, 10 μ M cobans, 5 μ M CoroNa Green, 5mM KCl, 130mM NaCl, 1mM MgCl ₂, 2mM CaCl ₂process cell with the 20mM HEPES damping fluid of 10mM glucose and make the sodion balance inside and outside cell, with concentration, be respectively again the sodion solution incubated cell of 10mM, 25mM, 55mM, 80mM, 100mM, 120mM, 130mM, in described sodion solution, also contain KCl, the 1mM MgCl of variable concentrations ₂, 2mM CaCl ₂, 10mM glucose and 20mM HEPES, wherein kalium ion and natrium ion concentration summation is 135mM.

10. the sodium potassium ion permeability based on TMRM as claimed in claim 5 compares value detection method, it is characterized in that, the gradient concentration potassium ion solution that adds CoroNa Green described in step D is to contain that 5 μ M CoroNa Green and potassium concentration are respectively 5,7.5,15,35,55,80, the solution of 130mM, also contains NaCl, the 1mM MgCl of variable concentrations in described solution ₂, 2mM CaCl ₂, 10mM glucose and 20mM HEPES, wherein kalium ion and natrium ion concentration summation is 135mM.