CN106191194A - A kind of detection method to intracellular reactive oxygen content - Google Patents
A kind of detection method to intracellular reactive oxygen content Download PDFInfo
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- CN106191194A CN106191194A CN201610499457.0A CN201610499457A CN106191194A CN 106191194 A CN106191194 A CN 106191194A CN 201610499457 A CN201610499457 A CN 201610499457A CN 106191194 A CN106191194 A CN 106191194A
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- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 230000003834 intracellular effect Effects 0.000 title claims abstract description 23
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims abstract description 20
- 229910052760 oxygen Inorganic materials 0.000 title claims abstract description 20
- 239000001301 oxygen Substances 0.000 title claims abstract description 20
- 239000003344 environmental pollutant Substances 0.000 claims abstract description 25
- 231100000719 pollutant Toxicity 0.000 claims abstract description 25
- 238000012360 testing method Methods 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 7
- 210000004027 cell Anatomy 0.000 claims description 76
- 101100063942 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) dot-1 gene Proteins 0.000 claims description 19
- 229910052785 arsenic Inorganic materials 0.000 claims description 9
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 claims description 9
- 239000006285 cell suspension Substances 0.000 claims description 8
- 239000007850 fluorescent dye Substances 0.000 claims description 8
- 238000011109 contamination Methods 0.000 claims description 7
- 230000005284 excitation Effects 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 7
- 230000012447 hatching Effects 0.000 claims description 6
- 230000029087 digestion Effects 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 210000002966 serum Anatomy 0.000 claims description 5
- 210000004369 blood Anatomy 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 4
- 238000010494 dissociation reaction Methods 0.000 claims description 4
- 230000005593 dissociations Effects 0.000 claims description 4
- 210000004691 chief cell of stomach Anatomy 0.000 claims description 2
- 238000004140 cleaning Methods 0.000 claims description 2
- 230000008859 change Effects 0.000 abstract description 7
- 238000000338 in vitro Methods 0.000 abstract description 4
- 231100000419 toxicity Toxicity 0.000 abstract description 3
- 230000001988 toxicity Effects 0.000 abstract description 3
- 230000006378 damage Effects 0.000 abstract description 2
- 238000005259 measurement Methods 0.000 abstract description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000004792 oxidative damage Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000004166 bioassay Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 231100000567 intoxicating Toxicity 0.000 description 1
- 230000002673 intoxicating effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention discloses a kind of detection method to intracellular reactive oxygen content, the method can obtain exposing under variable concentrations the DCF fluorescence signal value of single living cell corresponding to pollutant.The present invention is accurate to the detection method testing result of intracellular reactive oxygen content, overcome and cause cell quantity change so that the final inaccurate problem of fluorescence intensity measurement owing to pollutant expose, detection method can truly be reflected under certain concentration the individual cells fluorescence signal value of correspondence, it is thus possible to true and accurate reflection pollutant expose the damage to cell, lay the most sturdy basis for In vitro cell model application in pollutant toxicity assessment.
Description
Technical field
The present invention relates to a kind of detection method to intracellular reactive oxygen content, belong to biological activity cell detection field.
Background technology
In vitro cell model is a kind of at ex vivo situation Imitating biology growing environment, and detection environmental stimuli is to cell shadow
The technology rung.Use cell as subjects, have that sensitivity is high, can be greatly shortened the test period, mechanism of action is prone to
The advantage such as verifying, the efficiency not only increasing toxicology detection also reduces the use of animal, is weight in biological assessment system
The platform wanted.Oxidative damage mechanism is the important intoxicating mechanism of multiple pollutant.Pollutant are evaluated using In vitro cell model
During toxicity, it is often necessary to detect intracellular active oxygen (ROS) content to characterize the oxidative damage that body is subject to.Tradition
Intracellular ROS detection method by select suitable fluorescent probe cell is hatched, then by record cell sent out
The fluorescence signal value power penetrated determines intracellular ROS content.The fluorescence signal value of instrument record is sent by each cell
Fluorescence intensity and total cell number are determined.But in existing technology, usually ignore cell quantity change and caused
The change of whole fluorescence intensity, thus cause testing result inaccurate.In pollutant process-exposed, owing to pollutant effects can
Can cause viable count purpose difference between process group and matched group, it is therefore necessary to living cells quantity remaining to each hole is entered
Row is quantitatively to compensate the difference of the fluorescence signal value that cell quantity difference is caused.
Summary of the invention
The technical problem to be solved is to provide a kind of detection method to intracellular reactive oxygen content, the method
Can obtain exposing under variable concentrations the DCF fluorescence signal value of single living cell corresponding to pollutant.
For solving above-mentioned technical problem, the technical solution adopted in the present invention is as follows:
A kind of detection method to intracellular reactive oxygen content, comprises the steps:,
Step 1, selection HepG2 cell lines is as test cell strain, and digests test cell strain
Piping and druming process forms cell suspension；
Step 2, measures the cell density in cell suspension with blood counting chamber, and the density with 10000 cells in every hole will
Cell kind enters in 96 orifice plates；
Step 3, after planting plate 24h, carries out contamination and exposes, the exposure pollutant of variable concentrations are separately added into difference cell
Orifice plate in；
Step 4, after contamination 24h, uses 50 μ L DCF Incubating Solutions to replace, at 37 DEG C by culture medium original in each orifice plate
Hatch 25min, clean with PBS after hatching, use microplate reader to detect the DCF fluorescence signal value of each orifice plate inner cell；
Step 5, then add 100 μ L Red Dot 1 Incubating Solutions to each orifice plate, at 37 DEG C, hatch 15～30min, incubate
Wash with PBS after educating, use microplate reader to detect the Red Dot 1 fluorescence signal value of each orifice plate inner cell, obtain each
The quantity of living cells in orifice plate；
Step 6, the DCF fluorescence signal value in each orifice plate step 4 obtained is divided by the Red of cell in corresponding orifice plate
Dot 1 fluorescence signal value, obtains the DCF fluorescence signal value of corresponding single living cell under different exposure pollutant levels.
Wherein, in step 1, first test cell strain PBS is cleaned；Use trypsin-EDTA molten after cleaning
Liquid peptic cell；With the fresh culture (just preparation) containing serum (mass percentage concentration of serum is 10%) after cell dissociation
Terminate digestion；Finally the cell suspension obtained is blown even.
Wherein, in step 3, described exposure pollutant are arsenic.
Wherein, in step 4, the concentration of described DCF Incubating Solution is 10 μMs.
Wherein, in step 4, the excitation wavelength of the DCFH-DA fluorescent dye that described DCF Incubating Solution is corresponding is 485nm, launches
Wavelength is 530nm.
Wherein, in step 5, the concentration of described Red Dot 1 Incubating Solution is 5 μ g/ml.
Wherein, in step 5, the excitation wavelength of Red Dot 1 fluorescent dye that described Red Dot 1 Incubating Solution is corresponding is
630nm, a length of 710nm of transmitted wave.
Compared with existing detection method, the detection method of intracellular reactive oxygen content is had the advantage that by the present invention
For:
The present invention is accurate to the detection method testing result of intracellular reactive oxygen content, overcomes and leads owing to pollutant expose
Causing cell quantity change so that the final inaccurate problem of fluorescence intensity measurement, detection method can truly be reflected in
Individual cells fluorescence signal value corresponding under certain concentration such that it is able to true and accurate reflection pollutant expose the damage to cell
Wound, has laid the most sturdy basis for In vitro cell model application in pollutant toxicity assessment.
Accompanying drawing explanation
Fig. 1 is the number change figure of living cells in each orifice plate after the As of variable concentrations exposes；
Fig. 2 be variable concentrations As expose after each orifice plate inner cell DCF fluorescence signal value variation diagram；
Fig. 3 is each orifice plate inner cell DCF fluorescence letter under the variable concentrations As that the detection method of comparative example obtains exposes
Number value variation diagram.
Detailed description of the invention
Below in conjunction with the accompanying drawings technical solution of the present invention is described further.
Embodiment 1
A kind of detection method to intracellular reactive oxygen content, comprises the steps:
Step 1, selects HepG2 cell lines as test cell strain, examines under a microscope its growth conditions,
Remove original culture medium, clean cell 1～2 times with PBS, in culture dish, add 1mL trypsin-EDTA solutions,
Cell is digested；Treat that cell dissociation terminates, add the 10mL fresh culture containing serum and terminate digestion, the cell that will obtain
Suspension blows even；
Step 2, measures the cell density in cell suspension with blood counting chamber, and the density with 10000 cells in every hole will
Cell kind enters in 96 orifice plates；
Step 3, after planting plate 24h, carries out contamination and exposes, be separately added into not by the exposure pollutant arsenic of variable concentrations cell
In same orifice plate；
Step 4, utilizes DCFH-DA probe in detecting arsenic to expose the content of the lower intracellular reactive oxygen-derived free radicals (ROS) of induction, dye
After poison 24h, culture medium original in each orifice plate uses the MDCF Incubating Solution of 50 final concentration of 10 μ of μ L replace, incubates at 37 DEG C
Educate 25min, clean 2 times with PBS after hatching, use microplate reader to detect the DCF fluorescence signal value of each orifice plate inner cell；
Step 5, then in each orifice plate, add Red Dot 1 Incubating Solution of the final concentration of 5 μ g/ml of 100 μ L, at 37 DEG C
Hatch 15min, wash 2 times with PBS after hatching, use microplate reader to detect Red Dot 1 fluorescence of each orifice plate inner cell
Signal value, obtains the quantity of living cells in each orifice plate；
Step 6, the DCF fluorescence signal value in each orifice plate step 4 obtained is divided by the Red of cell in corresponding orifice plate
Dot 1 fluorescence signal value, obtains the fluorescence signal value of corresponding each living cells under different exposure pollutant levels.
The excitation wavelength of DCFH-DA fluorescent dye is 485nm, a length of 530nm of transmitted wave；Red Dot 1 fluorescent dye
Excitation wavelength is 630nm, a length of 710nm of transmitted wave.
As it is shown in figure 1, by Red Dot 1 fluorescence signal value it can be seen that along with exposing pollutant arsenic (As) concentration
Increasing, in each orifice plate, viable count amount occurs that significance reduces；Fig. 2 is each after Red Dot 1 fluorescence signal value standardization
The DCF signal value of orifice plate, figure it is seen that be in the of 5 μMs from exposing pollutant arsenic (As) concentration, exposes pollutant arsenic (As)
The increase of concentration significantly improves intracellular ROS content, and presents obvious dose-effect relationship.
Comparative example
A kind of detection method to intracellular reactive oxygen content, comprises the steps:
Step 1, selects HepG2 cell lines as test cell strain, examines under a microscope its growth conditions,
Remove original culture medium, clean cell 1～2 times with PBS, in culture dish, add 1mL trypsin-EDTA solutions,
Cell is digested；Treat that cell dissociation terminates, add the 10mL fresh culture containing serum and terminate digestion, the cell that will obtain
Suspension blows even；
Step 2, measures the cell density in cell suspension with blood counting chamber, and the density with 10000 cells in every hole will
Cell kind enters in 96 orifice plates；
Step 3, after planting plate 24h, carries out contamination and exposes, be separately added into not by the exposure pollutant arsenic of variable concentrations cell
In same orifice plate；
Step 4, after contamination 24h, uses the MDCF Incubating Solution of 50 final concentration of 10 μ of μ L by culture medium original in each orifice plate
Replace, at 37 DEG C, hatch 25min, clean 2 times with PBS after hatching；Microplate reader is used to detect each orifice plate inner cell
DCF fluorescence signal value.
The excitation wavelength of DCFH-DA fluorescent dye is 485nm, a length of 530nm of transmitted wave.
As shown in figures 2-3, without after Red Dot 1 fluorescence signal value standardized DCF fluorescence signal and standardization in
, process group when exposure pollutant arsenic (As) concentration of significant difference is 5 μMs should be there is and not show in existing a great difference: first
Significant difference, its reason is that the minimizing of cell quantity makes total DCF fluorescence signal value change inconspicuous；Secondly, Fig. 3's
Experimental result does not has obvious dose-effect relationship, and its reason have ignored cell quantity change equally and caused.
Claims (7)
1. the detection method to intracellular reactive oxygen content, it is characterised in that: comprise the steps:,
Step 1, selection HepG2 cell lines is as test cell strain, and test cell strain carries out digestion piping and druming
Process forms cell suspension；
Step 2, measures the cell density in cell suspension with blood counting chamber, with the density of 10000 cells in every hole by cell
Plant in 96 orifice plates；
Step 3, after planting plate 24h, carries out contamination and exposes cell, and the pollutant that expose of variable concentrations are separately added into different holes
In plate；
Step 4, after contamination 24h, uses culture medium original in each orifice plate 50 μ L DCF Incubating Solutions to replace, hatches at 37 DEG C
25min, cleans with PBS after hatching, and uses microplate reader to detect the DCF fluorescence signal value of each orifice plate inner cell；
Step 5, then add 100 μ L Red Dot 1 Incubating Solutions to each orifice plate, at 37 DEG C, hatch 15～30min, after hatching
Wash with PBS, use microplate reader to detect the Red Dot 1 fluorescence signal value of each orifice plate inner cell, obtain each orifice plate
The quantity of middle living cells；
Step 6, the DCF fluorescence signal value in each orifice plate step 4 obtained is divided by the Red Dot 1 of cell in corresponding orifice plate
Fluorescence signal value, obtains the DCF fluorescence signal value of corresponding single living cell under different exposure pollutant levels.
Detection method to intracellular reactive oxygen content the most according to claim 1, it is characterised in that: in step 1, first will
Test cell strain PBS cleans；Trypsin-EDTA solutions peptic cell is used after cleaning；With containing after cell dissociation
The culture medium of serum terminates digestion；Finally the cell suspension obtained is blown even.
Detection method to intracellular reactive oxygen content the most according to claim 1, it is characterised in that: in step 3, described
Exposure pollutant are arsenic.
Detection method to intracellular reactive oxygen content the most according to claim 1, it is characterised in that: in step 4, described
The concentration of DCF Incubating Solution is 10 μMs.
Detection method to intracellular reactive oxygen content the most according to claim 1, it is characterised in that: in step 4, described
The excitation wavelength of the DCFH-DA fluorescent dye that DCF Incubating Solution is corresponding is 485nm, a length of 530nm of transmitted wave.
Detection method to intracellular reactive oxygen content the most according to claim 1, it is characterised in that: in step 5, described
The concentration of Red Dot 1 Incubating Solution is 5 μ g/ml.
Detection method to intracellular reactive oxygen content the most according to claim 1, it is characterised in that: in step 5, described
The excitation wavelength of Red Dot 1 fluorescent dye that Red Dot 1 Incubating Solution is corresponding is 630nm, a length of 710nm of transmitted wave.
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| CN201710319298.6A CN107084957B (en) | 2016-06-29 | 2017-05-03 | Method for detecting content of active oxygen in cells |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107014789A (en) * | 2017-03-07 | 2017-08-04 | 闽南师范大学 | The detection method of active oxygen inside and outside a kind of Phytoplankton Cells |
| CN107084957A (en) * | 2016-06-29 | 2017-08-22 | 南京大学 | A kind of method detected to intracellular reactive oxygen content |
| CN112326607A (en) * | 2020-10-16 | 2021-02-05 | 暨南大学 | Low-concentration ROS detection method and application thereof |
| CN114200065A (en) * | 2021-12-08 | 2022-03-18 | 维塔探索(广东)科技有限公司 | Method for quantitatively measuring content of in vivo/in vitro superoxide radical generated by environmental stress |
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| US20060276385A1 (en) * | 2003-01-13 | 2006-12-07 | Hanjoong Jo | Anti-inflammatory agents and methods of their use |
| CN1818620A (en) * | 2006-03-14 | 2006-08-16 | 浙江大学 | Improved method for determinating intracellular active oxygen |
| CN1818621A (en) * | 2006-03-14 | 2006-08-16 | 浙江大学 | Improvement for intracellular ROS inspecting sensitivity |
| TWI448687B (en) * | 2008-05-06 | 2014-08-11 | Colgate Palmolive Co | Method of measuring effects of compounds on cell reactive oxygen species production |
| CN101671722A (en) * | 2009-09-28 | 2010-03-17 | 华东理工大学 | Evaluation method of cell biology safety of silicon dioxide nanoparticle |
| CN102735806A (en) * | 2011-04-08 | 2012-10-17 | 复旦大学 | A method for screening traditional Chinese medicine monomers |
| CN103342720B (en) * | 2013-07-01 | 2015-08-26 | 南京大学 | The hydroxyl radical free radical probe of near infrared BODIPY class and preparation method and use thereof |
| CN103472046A (en) * | 2013-09-25 | 2013-12-25 | 山东理工大学 | Method of analyzing heavy metal pollution of vegetable based on reactive oxygen species level |
| CN106191194A (en) * | 2016-06-29 | 2016-12-07 | 南京大学 | A kind of detection method to intracellular reactive oxygen content |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107084957A (en) * | 2016-06-29 | 2017-08-22 | 南京大学 | A kind of method detected to intracellular reactive oxygen content |
| CN107084957B (en) * | 2016-06-29 | 2020-09-08 | 南京大学 | Method for detecting content of active oxygen in cells |
| CN107014789A (en) * | 2017-03-07 | 2017-08-04 | 闽南师范大学 | The detection method of active oxygen inside and outside a kind of Phytoplankton Cells |
| CN107014789B (en) * | 2017-03-07 | 2019-07-23 | 闽南师范大学 | The detection method of active oxygen inside and outside a kind of Phytoplankton Cells |
| CN112326607A (en) * | 2020-10-16 | 2021-02-05 | 暨南大学 | Low-concentration ROS detection method and application thereof |
| CN114200065A (en) * | 2021-12-08 | 2022-03-18 | 维塔探索(广东)科技有限公司 | Method for quantitatively measuring content of in vivo/in vitro superoxide radical generated by environmental stress |
| CN114200065B (en) * | 2021-12-08 | 2022-07-26 | 维塔探索(广东)科技有限公司 | Method for quantitatively measuring content of in vivo/in vitro superoxide radical generated by environmental stress |
| CN114295596A (en) * | 2021-12-30 | 2022-04-08 | 无锡代际生物科技有限公司 | Sperm quality multi-parameter detection kit |
| CN114295596B (en) * | 2021-12-30 | 2024-04-05 | 无锡代际生物科技有限公司 | Sperm quality multiparameter detection kit |
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| CN107084957B (en) | 2020-09-08 |
| CN107084957A (en) | 2017-08-22 |
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