CN107502649A - A kind of new quick stain for cell and its application - Google Patents
A kind of new quick stain for cell and its application Download PDFInfo
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- CN107502649A CN107502649A CN201710879051.XA CN201710879051A CN107502649A CN 107502649 A CN107502649 A CN 107502649A CN 201710879051 A CN201710879051 A CN 201710879051A CN 107502649 A CN107502649 A CN 107502649A
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Abstract
It can be the aqueous solution that the present invention, which provides a kind of new quick stain for cell and its application, the stain for cell, and it includes solute toluidine blue and sodium sulphate.Include its directly contacting dyeing with cell or tissue to be contaminated using the method for the stain for cell, complete the dyeing to cell.Stain for cell of the present invention have rapid dyeing and dye cell advantage not easy to fade.In addition, stain for cell of the present invention also has the advantages that safety and environmental protection, composition are simple, dosage is few, price is low, preparation is convenient, can stored for a long time；Furthermore when the cell to being attached on microcarrier dyes, the Color of stain for cell of the present invention is better than ordinary stain agent, it is clear to show as cell color, and microcarrier is not colored；In addition, the more conventional mast cell coloring agent of stain for cell of the present invention is faster, living cells can be dyed, the coloring to particle is more sensitive, apparent.
Description
Technical field
The present invention relates to a kind of stain for cell and its application, more particularly to a kind of quick stain for cell and its application,
Belong to cell dyeing field.
Background technology
The cellular morphology details being frequently necessary in basic research in observation experiment, the fine knot of cell can be made by dyeing
Structure shows different colors, so as to can clearly show cell image under light microscopic.The colouring method step that prior art provides
Rapid more, time length, and need by a series of programs such as cell fixations, it is normal in order in time, rapidly observe cellular morphology. more
Using fast staining, but the current following defect of fast staining generally existing：
(1) reagent used in rapid dyeing agent has potential pollution or harm mostly to environment or human body.
(2) dye liquor complicated component, trouble is prepared, needs mostly now with the current, can not be preserved for a long time.
(3) the use process of coloring agent is comparatively laborious, need to fix cell, dyeing course time length.
(4) narrow application range, normally only just there is the effect of fast dye to particular kind of cell.
(5) dyeing time is short, after rapid dyeing, fades in a short period of time, can not meet to see in real time for a long time
Examine the needs of living cells.
In summary, also there is many defects in the quick living cells coloring agent that prior art provides, therefore, this area is needed badly
A kind of new quick living cells coloring agent of exploitation, to overcome the one or more in drawbacks described above.
The content of the invention
Needed in view of reality, it is a primary object of the present invention to provide a kind of cell dyeing composition, the cell dyeing
Composition can be configured to stain for cell, and the coloring agent can make living cells rapid dyeing, and make by dye cell very long
It is colour-fast in a period of time, the needs in the form of meeting long-time Real Time Observation living cells.
It is still another object of the present invention to provide the stain for cell for preparing to obtain by aforementioned cells colouring compositions.
It is still another object of the present invention to provide the application of aforementioned cells colouring compositions or aforementioned cells coloring agent.
To achieve the above object, the present invention provides a kind of cell dyeing composition, wherein, the cell dyeing composition bag
Include toluidine blue and sodium sulphate.
Aforementioned cells colouring compositions of the present invention can be configured to cell dyeing liquid with water, by the toluidine blue in composition and
Sodium sulphate is dissolved in water.Experiments show that when carrying out living cells rapid dyeing, toluidine blue
(Toluidine blue) has vivid coloring effect, and cell color is quickly, it is only necessary to and 2 seconds or so, but colour after shorter
In time, cell starts to fade after about 15min, can not meet the needs of long-time Real Time Observation living cells form.The present invention
The combination of toluidine blue and sodium sulphate, draws by testing repeatedly, and inventor has found to add in Toluidine blue staining liquid
Sodium sulphate can significantly extend by the fading time of dye cell, have the function that dying-stable, lasting, cell color is after very
All without colour fading in long a period of time, it can maintain cell color as long as a few days (up to 48h) after mounting.In addition, as sample is dried
Cell fades afterwards, and cell shows bright-coloured color and luster again if this dyeing liquor is added dropwise again.
Toluidine blue (Toluidine Blue, CAS numberings of the present invention：6586-04-5) it is bottle green or black gray expandable
Powder, its is commercially available, and the blue metering of Toluene amine of the present invention is calculated with net content.
Sodium sulphate of the present invention may be either that hydrated form is alternatively anhydrous form, and the metering of the following sodium sulphate of the present invention is equal
Calculated with anhydrous form.
As a concrete form of aforementioned cells colouring compositions of the present invention, with the gross weight of the cell dyeing composition
For 100% meter, the mass percent of the toluidine blue is 1.6%~14.3%.
As a concrete form of aforementioned cells colouring compositions of the present invention, with the gross weight of the cell dyeing composition
For 100% meter, the mass percent of the sodium sulphate is 85.7%~98.4%.
As a concrete form of aforementioned cells colouring compositions of the present invention, with the gross weight of the cell dyeing composition
For 100% meter, the mass percent of the toluidine blue is 1.6%~14.3%, and the mass percent of the sodium sulphate is
85.7%~98.4%.
Foregoing of the present invention can have in effective sale other substitute title, such as kit, external member, set group or
System etc..Two kinds of materials may be mixed together packaging in foregoing, also can independently separate packaging.
Term used herein " comprising " means that composition or method include cited key element, but is not precluded from other and wants
Element.When " substantially or being mainly made up of some components or step " is used for combinations of definitions thing and method, should mean to exclude to group
Compound or method have the other element of any essential meaning." being made up of component or step " should mean exclusion be directed to opinion group
It is more than the other compositions of trace elements for compound and substantial method steps.As defined in each of these transitional terms
Embodiment is within the scope of the present invention.Therefore, method and composition may be intended to include other steps and component (including) or another
Selection is to include unessential step and composition (substantially or be mainly made from it or step) or another selection only to mean
Methods described step or composition (being made from it).
All numerical value of the present invention are specified and (such as temperature, time, concentration and weight etc., include the scope of each of which) logical
Suitably change often but with 0.1 or 1.0 increment the approximation of (+) or (-).All numerical value, which are specified, can be regarded as above having art
Language " about ".
On the other hand, the present invention provides a kind of stain for cell, wherein, the stain for cell is the aqueous solution, and it includes molten
Matter toluidine blue and solute sodium sulphate.
The stain for cell can be formulated using above-mentioned cell dyeing composition.As it was previously stated, the stain for cell has
Have rapid dyeing and dye cell advantage not easy to fade.In addition, stain for cell of the present invention also has safety and environmental protection, composition
Simply, dosage is few, price is low, preparation is convenient, it is excellent to store more than 1 year (existing in general dye liquor is all matching while using) etc. for a long time
Point.
As an embodiment of aforementioned cells coloring agent, the mass concentration scope of the solute toluidine blue is
0.01mg/ml~5mg/ml.
As an embodiment of aforementioned cells coloring agent, the mass concentration scope of the solute sodium sulphate is
0.615mg/ml~30mg/ml.
As an embodiment of aforementioned cells coloring agent, the mass concentration scope of the solute toluidine blue is
0.01mg/ml~5mg/ml, the mass concentration scope of the solute sodium sulphate is 0.615mg/ml~30mg/ml.
Another further aspect, the present invention provide foregoing cell dyeing composition or foregoing stain for cell to cell or group
Knit the application in dyeing.
In practical application aforementioned cells colouring compositions, aforementioned cells coloring agent can be first configured to, and for thin
Born of the same parents' coloring agent, is directly applied.
In practical application, the cell on cell to be contaminated, microcarrier or the cell on Tu piece can be contaminated with cell of the present invention
Toner directly contacts dyeing, you can cell is being contaminated.Dyeing time typically only needs 0.5~2min to complete, and fades
Time can maintain a couple of days.Using stain for cell of the present invention, dying operation is simple and efficient, and cell, only need to be by cell without fixed
Directly mixed with dye liquor, after several minutes can clearly dye cell, the Real Time Observation being very beneficial in cytomorphology research.
Therefore, the invention provides a kind of method of cell dyeing, it comprises the following steps：
(1) aforementioned cells colouring compositions are configured to the aqueous solution：
(2) step (1) resulting solution or foregoing stain for cell are directly contacted into dyeing with cell or tissue to be contaminated, it is excellent
Choosing 0.5~2min of dyeing, completes the dyeing to cell；
Preferably, the concentration range of toluidine blue is 0.01mg/ml~5mg/ described in obtained aqueous solution in step (1)
Ml, the concentration range of the sodium sulphate is 0.615mg/ml~30mg/ml.
This colouring method is applied widely, is suitable for almost the external dyeing of all individual cells.The species of cell includes
But melanoma cells, cervical carcinoma, liver cancer, onion endothelial cell, mast cell, mouth epithelial cells, lung are not limited into fiber
Cell or monocytes/macrophages etc..The cell that the present invention is colored includes but is not limited to suspension cell, attached cell, microcarrier
On cell or Tu piece on cell.
Present invention cell to be contaminated can be living cells.It should be noted that the stain for cell of the present invention may be equally applied to
Dead cell.The dyeing the present invention is especially suitable for living cells is simply said, because Real Time Observation can be carried out to its cellular morphology.
Tissue of the present invention can be plant tissue.
It is of the invention to find in the specific implementation in the dyeing to microcarrier culture cell, the dye of stain for cell of the present invention
Color effect is better than ordinary stain agent, and such as giemsa (Jim Sa), eosin (Yihong) coloring agent, this is primarily due to use this hair
Bright coloring agent can clearly show eucaryotic cell structure, but not dye microcarrier, be more beneficial for the observation of staining cell；And giemsa makes
Microcarrier makes microcarrier be in orange red in pale red, eosin, it is difficult to which microcarrier after dyeing and cell differentiation are come, is unfavorable for
The observation of staining cell.Therefore, this colouring method can be used for the form of home position observation microcarrier culture cell.From the above it can be seen that this
Invention provides a kind of method of the form of home position observation microcarrier culture cell, and it comprises the following steps：
(1) microcarrier culture cell is dyed using the method for aforementioned cells dyeing；
(2) home position observation is by the form of the microcarrier culture cell of dye.
The present invention in the specific implementation find toluidine blue dye there is metachromasia, can dye mast cell in enrich it is thermophilic
Alkali grain, faster using the more traditional mast cell dyeing of coloring agent of the present invention, the coloring to particle is more sensitive, apparent.
And during traditional mast cell Toluidine blue staining, multiple rings such as cell needs to centrifuge, dries, fixes, dyeing, being dehydrated
Section, whole time about need 2.5~3 hours.Therefore, this method is alternatively arranged as inducing differentiation cultured mast cells process in vitro
In, the morphological method of realtime qualification mast cell maturity.It is from the above it can be seen that loose thin the invention provides a kind of realtime qualification
The morphological method of born of the same parents' maturity, it comprises the following steps：
(1) basophilic stippling in mast cell is dyed using the method for aforementioned cells dyeing；
(2) staining conditions of basophilic stippling are observed to determine the maturity of mast cell.
In summary, invention broadly provides a kind of quick stain for cell and its application, it has the following advantages that：
(1) dye liquor is made up of toluidine blue and sodium sulphate and distilled water.Toluidine blue is medical, and human body is pacified
Entirely, and do not pollute the environment；Also nonhazardous acts on sodium sulphate.Therefore the dye liquor is safe and non-toxic pollution-free.
(2) dye liquor composition is few, is common agents, and cost is low (to press current cost of material, the reagent of 100mL dyeing liquors is former
Material cost only about needs 2 yuan), it is convenient to prepare, and can deposit for a long time, without matching while using.
(3) dying operation is simple and efficient, and cell only need to directly mix cell without fixed with dye liquor, after several minutes i.e.
Can clearly dye cell, and dying-stable, persistently, the Real Time Observation being very beneficial in cytomorphology research.
(4) this colouring method is applied widely, is suitable for almost the external dyeing of all individual cells.
(5) ordinary stain agent (such as giemsa, eosin) is better than to the Color of microcarrier culture cell, because of the dyeing
Method can clearly show eucaryotic cell structure, but not dye microcarrier；And giemsa makes microcarrier make micro- load in pale red, eosin
Body is in orange red, so as to influence the definition of cell dyeing.Therefore, it is thin to be used for home position observation microcarrier culture for this colouring method
The form of born of the same parents.
(6) toluidine blue dye has metachromasia, can dye mast cell in enrich basophilic stippling, this method compared with
Faster toward mast cell dyeing, the coloring to particle is more sensitive, apparent, and therefore, or Differentiation Induction in vitro culture is loose
In cell processes, realtime qualification mast cell maturity easy-to-use morphological method.
Brief description of the drawings
Fig. 1 is the result figure of dyeing of the gained fast dye liquor of toluidine blue to cells on microcarriers in the embodiment of the present invention 1.
Fig. 2 is the fast dye liquor of toluidine blue obtained by the embodiment of the present invention 1 to the result figure of mouth epithelial cells smear staining.
Fig. 3 is that the gained fast dye liquor of toluidine blue dyes under cell moisture state to mast cell in the embodiment of the present invention 1
Result figure.
Fig. 4 is the knot that the fast dye liquor of gained toluidine blue dyes to mast cell in the dry state in the embodiment of the present invention 1
Fruit is schemed.
Fig. 5 is the result figure that the gained fast dye liquor of toluidine blue dyes to onion endothelial cell in the embodiment of the present invention 1.
Fig. 6 is the B16 melanoma cells being inoculated in comparative example 1 on microcarrier, using Giemsa dye liquor rapid dyeings
Result figure.
Fig. 7 is the B16 melanoma cells being inoculated in comparative example 1 on microcarrier, using Eosin dye liquor rapid dyeings
Result figure.
Embodiment
In order to which technical characteristic, purpose and the beneficial effect of the present invention is more clearly understood, in conjunction with specific implementation
Example carries out described further below to technical scheme, it should be understood that these examples are merely to illustrate the present invention rather than limit
The scope of the present invention processed.In embodiment, each Starting reagents material is commercially available, and the experimental method of unreceipted actual conditions is
Conventional method known to art and normal condition, or according to the condition proposed by apparatus manufacturer.
Embodiment 1
The present embodiment provides a kind of rapid dyeing agent and its application, the rapid dyeing agent and is prepared as follows obtaining：
By 225mg toluidine blues (mass percent 10%) and 2025mgNa2SO4(mass percent 90%) is dissolved in steaming
Distilled water, after solute is completely dissolved, it is settled to 100ml with distilled water and stands overnight, as the present embodiment rapid dyeing agent-first
The fast dye liquor of aniline blue.The present embodiment tests the Color of the gained fast dye liquor of toluidine blue as follows：
(1) dyeing of cells on microcarriers
B-16 melanoma cells are inoculated on microcarrier, and add medium culture.With preceding from microcarrier culture body
System takes the microcarrier cell suspension containing B-16 melanoma cells and each drop of the fast dye liquor of gained toluidine blue to be placed in slide
On, mix, dye 0.5~2min, while staining conditions are observed under the microscope, in good time direct mounting.Then in light microscopic (40 ×
Camera lens) under observe.Acquired results are as shown in figure 1, the B-16 melanoma cells being as can be seen from Figure 1 inoculated with microcarrier
Distinct aubergine (arrow is signified), clear in structure, microcarrier is not colored；Apoptotic cell karyopycnosis contaminates deeply, and how swollen non-viable non-apoptotic cell is
Greatly, karyolysis or fragmentation；But the cell image for being hidden in microcarrier deep is relatively fuzzy, after dyeing mounting in 48h, connect on microcarrier
The B-16 melanoma cells of kind remain the aubergine of distinctness, and clear in structure, microcarrier is not colored.
(2) mouth epithelial cells smear staining
A small amount of mouth epithelial cells are scraped, are placed on slide, adds the gained fast dye liquor one of toluidine blue to drip, mixes, dyeing
0.5~2min, while staining conditions are observed under the microscope, in good time direct mounting.Then observed under light microscopic (40 × camera lens).
Acquired results as shown in Fig. 2 as can be seen from Figure 2 mouth epithelial cells can clearly dye, cellular morphology is clear, dye mounting
Afterwards in 48h, it is clear that mouth epithelial cells remain clear dye, cellular morphology.
(3) mast cell dyes
(3.1) mouse femur bone marrow cell is taken, through conditioned medium culture surrounding, induces differentiation into mast cell (bone
Marrow-derived mast cells, BMMC, represent that mast cell is ripe full of particle in endochylema), it is standby.
(3.2) mast cell suspension obtained by appropriate step (3.1) is taken 200rpm, 1min, to be taken advantage of in rejection tablet on centrifugation smearing machine
The μ l of the fast dye liquor of gained toluidine blue about 100 are added dropwise in cell immediately when moistening.
(3.3) 10~15min of dye, dyeing observes staining conditions under the microscope simultaneously, in good time directly mounting, terminates dye
Color, unnecessary dye liquor is sucked along around cover glass with paper handkerchief.
(3.4) in 100 × oily Microscopic observation.
Acquired results are as shown in figure 3, from figure 3, it can be seen that step (3.1)~step (3.4) can be made in mast cell
Particle it is clear dye (it is particle in endochylema that arrow is signified in Fig. 3), but can not dye nucleus, dye 48h after mounting
Interior, the particle in mast cell remains clear dye, but can not dye nucleus., will be thin after rejection tablet if nucleus need to be observed
Then the μ l of dye liquor 100 are added dropwise in drying at room temperature 15min in born of the same parents' smear, repeat step (3.3) and (3.4), acquired results such as Fig. 4 institutes
Show, as can be seen from Figure 4 under drying regime the fast dye liquor not only can the thin arrow of dye it is signified be particle (Fig. 4 in endochylema
In thin arrow it is signified), moreover it is possible to the dye (block arrow is signified in Fig. 4) that makes nucleus clear, dye after mounting in 48h, nucleus is protected
Hold clear dye.In addition, comparison diagram 3 and Fig. 4 have been also found that under cell moisture state to BMMC cytoplasmic granules dye compared with
More sensitive under cell drying regime, particle shows sharper keen and clear.
(4) onion endothelial cell dyes
(4.1) appropriate onion endothelium is stripped, is laid on slide.
(4.2) with alcolhol burner flame it is careful, it is of short duration heating the slide bottom several seconds, make onion endothelium moderately dry.
(4.3) the gained fast dye liquor of toluidine blue is added dropwise, 5~10min of dye, under the microscope dyeing while observation dyeing feelings
Condition, directly mounting, termination dyeing, unnecessary dye liquor is sucked along around cover glass with paper handkerchief in good time.
(4.4) observed under light microscopic (40 × camera lens).
Acquired results are as shown in figure 5, as can be seen from Figure 5 the eucaryotic cell structure of onion endothelial cell is high-visible, cell
Core dyes blue-green (arrow is signified in Fig. 5) by clear, dyes after mounting in 48h, the eucaryotic cell structure of onion endothelial cell is protected
Hold high-visible, nucleus dyes blue-green by clear.
Embodiment 2
The present embodiment provides a kind of rapid dyeing agent and its application, the rapid dyeing agent and is prepared as follows obtaining：
By 50mg toluidine blues (mass percent 5%) and 950mgNa2SO4(mass percent 95%) is dissolved in distillation
Water, after solute is completely dissolved, 100ml is settled to distilled water, is stood overnight, as the present embodiment rapid dyeing agent-toluene
The blue fast dye liquor of amine.
(1) dyeing of cells on microcarriers
B-16 melanoma cells are inoculated on microcarrier, and add medium culture.With preceding from microcarrier culture body
System takes the microcarrier cell suspension containing B-16 melanoma cells and each drop of the fast dye liquor of gained toluidine blue to be placed in slide
On, mix, dye 0.5~2min, while staining conditions are observed under the microscope, in good time direct mounting.Result of the test is shown：It is micro-
The B-16 melanoma cells being inoculated with carrier the aubergine of distinctness, and clear in structure, microcarrier is not colored, after dyeing mounting
In 48h, the B-16 melanoma cells being inoculated with microcarrier remain the aubergine of distinctness, and clear in structure, microcarrier is not colored.
(2) mouth epithelial cells smear staining
A small amount of mouth epithelial cells are scraped, are placed on slide, adds the gained fast dye liquor one of toluidine blue to drip, mixes, dyeing
0.5~2min, while staining conditions are observed under the microscope, in good time direct mounting.Result of the test is shown：Mouth epithelial cells can
Clear dye, cellular morphology is clear, dyes after mounting in 48h, and it is clear that mouth epithelial cells remain clear dye, cellular morphology
It is clear.
(3) mast cell dyes
(3.1) mouse femur bone marrow cell is taken, through conditioned medium culture surrounding, induces differentiation into mast cell (bone
Marrow-derived mast cells, BMMC, represent that mast cell is ripe full of particle in endochylema), it is standby.
(3.2) mast cell suspension obtained by appropriate step (3.1) is taken 200rpm, 1min, to be taken advantage of in rejection tablet on centrifugation smearing machine
The μ l of the fast dye liquor of gained toluidine blue about 100 are added dropwise in cell immediately when moistening.
(3.3) 10~15min of dye, dyeing observes staining conditions under the microscope simultaneously, in good time directly mounting, terminates dye
Color, unnecessary dye liquor is sucked along around cover glass with paper handkerchief.
(3.4) in 100 × oily Microscopic observation.
Microscopy results are shown：The present embodiment dye liquor particle in mast cell can be made clear dye, but can not
Contaminate nucleus, dye after mounting in 48h, the particle in mast cell keeps clear dye, but can not dye nucleus.If need to see
Examine nucleus, by cell smear in drying at room temperature 15min after rejection tablet, be then added dropwise the μ l of dye liquor 100, repeat step (3.3) and
(3.4), you can it was observed that under drying regime the fast dye liquor not only can the particle in dye cytoplasm, moreover it is possible to make nucleus clear
Dye, dye after mounting in 48h, nucleus remains clear dye.In addition, to BMMC cytoplasmic granules under cell moisture state
Dye it is more sensitive compared with cell drying regime, particle shows sharper keen and clear.
(4) onion endothelial cell dyes
(4.1) appropriate onion endothelium is stripped, is laid on slide.
(4.2) with alcolhol burner flame it is careful, it is of short duration heating the slide bottom several seconds, make onion endothelium moderately dry.
(4.3) the gained fast dye liquor of toluidine blue is added dropwise, 5~10min of dye, under the microscope dyeing while observation dyeing feelings
Condition, directly mounting, termination dyeing, unnecessary dye liquor is sucked along around cover glass with paper handkerchief in good time.
(4.4) observed under light microscopic (40 × camera lens).
Microscopy results are shown：The eucaryotic cell structure of onion endothelial cell is high-visible, and nucleus is dyed by clear
Blue-green, dye after mounting in 48h, the eucaryotic cell structure of onion endothelial cell remains high-visible, and nucleus is by clear dye
Into blue-green.
Embodiment 3
The present embodiment provides a kind of rapid dyeing agent and its application, the rapid dyeing agent and is prepared as follows obtaining：
By 400mg toluidine blues (mass percent 14%) and 2457mgNa2SO4 (mass percent 86%) is dissolved in steaming
Distilled water, after solute is completely dissolved, 100ml is settled to distilled water, is stood overnight, as the present embodiment rapid dyeing agent-first
The fast dye liquor of aniline blue.
(1) dyeing of cells on microcarriers
B-16 melanoma cells are inoculated on microcarrier, and add medium culture.With preceding from microcarrier culture body
System takes the microcarrier cell suspension containing B-16 melanoma cells and each drop of the fast dye liquor of gained toluidine blue to be placed in slide
On, mix, dye 0.5~2min, while staining conditions are observed under the microscope, in good time direct mounting.Result of the test is shown：It is micro-
The B-16 melanoma cells being inoculated with carrier the aubergine of distinctness, and clear in structure, microcarrier is not colored, after dyeing mounting
In 48h, the B-16 melanoma cells being inoculated with microcarrier remain the aubergine of distinctness, and clear in structure, microcarrier is not colored.
(2) mouth epithelial cells smear staining
A small amount of mouth epithelial cells are scraped, are placed on slide, adds the gained fast dye liquor one of toluidine blue to drip, mixes, dyeing
0.5~2min, while staining conditions are observed under the microscope, in good time direct mounting.Result of the test is shown：Mouth epithelial cells can
Clear dye, cellular morphology is clear, dyes after mounting in 48h, and it is clear that mouth epithelial cells remain clear dye, cellular morphology
It is clear.
(3) mast cell dyes
(3.1) mouse femur bone marrow cell is taken, through conditioned medium culture surrounding, induces differentiation into mast cell (bone
Marrow-derived mast cells, BMMC, represent that mast cell is ripe full of particle in endochylema), it is standby.
(3.2) mast cell suspension obtained by appropriate step (3.1) is taken 200rpm, 1min, to be taken advantage of in rejection tablet on centrifugation smearing machine
The μ l of the fast dye liquor of gained toluidine blue about 100 are added dropwise in cell immediately when moistening.
(3.3) 10~15min of dye, dyeing observes staining conditions under the microscope simultaneously, in good time directly mounting, terminates dye
Color, unnecessary dye liquor is sucked along around cover glass with paper handkerchief.
(3.4) in 100 × oily Microscopic observation.
Microscopy results are shown：The present embodiment dye liquor particle in mast cell can be made clear dye, but can not
Contaminate nucleus, dye after mounting in 48h, the particle in mast cell remains clear dye, but can not dye nucleus.If need
Observe nucleus, by cell smear in drying at room temperature 15min after rejection tablet, be then added dropwise the μ l of dye liquor 100, repeat step (3.3) and
(3.4), you can it was observed that under drying regime the fast dye liquor not only can the particle in dye endochylema, moreover it is possible to make nucleus clear
Dye, dye after mounting in 48h, nucleus remains clear dye.In addition, to BMMC cytoplasmic granules under cell moisture state
Dye it is more sensitive compared with cell drying regime, particle shows sharper keen and clear.
(4) onion endothelial cell dyes
(4.1) appropriate onion endothelium is stripped, is laid on slide.
(4.2) with alcolhol burner flame it is careful, it is of short duration heating the slide bottom several seconds, make onion endothelium moderately dry.
(4.3) the gained fast dye liquor of toluidine blue is added dropwise, 5~10min of dye, under the microscope dyeing while observation dyeing feelings
Condition, directly mounting, termination dyeing, unnecessary dye liquor is sucked along around cover glass with paper handkerchief in good time.
(4.4) observed under light microscopic (40 × camera lens).
Microscopy results are shown：The eucaryotic cell structure of onion endothelial cell is high-visible, and nucleus is dyed by clear
Blue-green, dye after mounting in 48h, the eucaryotic cell structure of onion endothelial cell remains high-visible, and nucleus is by clear dye
Into blue-green.
Comparative example
For the dyeing of the cells on microcarriers in embodiment 1~3, B-16 melanoma cells are inoculated in by this comparative example
On identical microcarrier, the Giemsa dye liquors rapid dyeing or Eosin dye liquor rapid dyeings that are provided using prior art, dyeing knot
Respectively as shown in FIG. 6 and 7, it can be seen that Giemsa dye liquors make microcarrier be in pale red from Fig. 6 and Fig. 7, Eosin contaminates fruit figure
Liquid makes microcarrier be in orange red, is unfavorable for coming microcarrier and cell differentiation, is unfavorable for the observation of staining cell.Finally say
Bright is：Above example is merely to illustrate the implementation process and feature of the present invention, and the technical scheme being not intended to limit the present invention is most
The present invention is described in detail with reference to above-described embodiment for pipe, it will be understood by those within the art that：Still can be with
The present invention is modified or equivalent substitution, any modification or partial replacement without departing from the spirit and scope of the present invention,
It all should cover among protection scope of the present invention.
Claims (10)
1. a kind of cell dyeing composition, wherein, the cell dyeing composition includes toluidine blue and sodium sulphate.
2. cell dyeing composition according to claim 1, wherein, using the gross weight of the cell dyeing composition as
100% meter, the mass percent of the toluidine blue is 1.6%~14.3%.
3. cell dyeing composition according to claim 1 or 2, wherein, with the gross weight of the cell dyeing composition
For 100% meter, the mass percent of the sodium sulphate is 85.7%~98.4%.
4. a kind of stain for cell, wherein, the stain for cell is the aqueous solution, and it includes solute toluidine blue and sodium sulphate.
5. stain for cell according to claim 4, wherein, the mass concentration scope of the solute toluidine blue is
0.01mg/ml~5mg/ml.
6. the stain for cell according to claim 4 or 5, wherein, the mass concentration scope of the sodium sulphate is
0.615mg/ml~30mg/ml.
It is 7. thin any one of cell dyeing composition according to any one of claims 1 to 3 or claim 4~6
Application of born of the same parents' coloring agent in being dyed to cell or tissue.
8. a kind of method of cell dyeing, methods described comprise the following steps：
(1) cell dyeing composition according to any one of claims 1 to 3 is configured to the aqueous solution；
(2) by the stain for cell any one of step (1) resulting solution or claim 4~6 and cell to be contaminated or
Tissue directly contact dyeing, preferably dyes 0.5~2min, completes the dyeing to cell；
Preferably, the mass concentration scope of toluidine blue is 0.01mg/ml~5mg/ described in obtained aqueous solution in step (1)
Ml, the mass concentration scope of the sodium sulphate is 0.615mg/ml~30mg/ml；
Preferably, it is thin that the species for the cell being colored includes melanoma cells, cervical carcinoma, liver cancer, onion endothelial cell, hypertrophy
Born of the same parents, mouth epithelial cells, lung fibroblast or monocytes/macrophages；
Preferably, the cell being colored includes suspension cell, attached cell, the cell on microcarrier or the cell on Tu piece, institute
That states is organized as plant tissue；
Preferably, the cell being colored is living cells.
9. a kind of method of the form of home position observation microcarrier culture cell, it comprises the following steps：
(1) microcarrier culture cell is dyed using the method for cell dyeing described in claim 8；
(2) home position observation is by the form of the microcarrier culture cell of dye.
10. a kind of morphological method of realtime qualification mast cell maturity, it comprises the following steps：
(1) basophilic stippling in mast cell is dyed using the method for cell dyeing described in claim 8；
(2) staining conditions of basophilic stippling are observed to determine the maturity of mast cell.
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| CN111748218A (en) * | 2020-07-06 | 2020-10-09 | 陕西中医药大学 | Coloring agent for detecting microorganisms in water |
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| CN1987402A (en) * | 2006-09-09 | 2007-06-27 | 王晓银 | Multifunction quick dyeing liquid preparation and use for chest ascitic fluid and gynecologic exfoliated cell |
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| CN105842037A (en) * | 2016-03-21 | 2016-08-10 | 山东农业大学 | Staining method for simultaneously displaying mast cells and acidophilic cells |
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| CN1987402A (en) * | 2006-09-09 | 2007-06-27 | 王晓银 | Multifunction quick dyeing liquid preparation and use for chest ascitic fluid and gynecologic exfoliated cell |
| CN105004714A (en) * | 2015-06-10 | 2015-10-28 | 苏州东辰林达检测技术有限公司 | Detection reagent for nitrate in hot pickled tuber mustard and detection method thereof |
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| CN111748218A (en) * | 2020-07-06 | 2020-10-09 | 陕西中医药大学 | Coloring agent for detecting microorganisms in water |
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