CN106092991A - A kind of utilize DAPI fluorescent dye to the detection method of active o content in living cells - Google Patents
A kind of utilize DAPI fluorescent dye to the detection method of active o content in living cells Download PDFInfo
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- CN106092991A CN106092991A CN201610503947.3A CN201610503947A CN106092991A CN 106092991 A CN106092991 A CN 106092991A CN 201610503947 A CN201610503947 A CN 201610503947A CN 106092991 A CN106092991 A CN 106092991A
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- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 title claims abstract description 34
- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 23
- 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 8
- 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
- 239000001963 growth medium Substances 0.000 claims description 8
- 238000011109 contamination Methods 0.000 claims description 7
- 230000005284 excitation Effects 0.000 claims description 7
- 230000012447 hatching Effects 0.000 claims description 7
- 230000029087 digestion Effects 0.000 claims description 5
- 230000008569 process Effects 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
- 210000002966 serum Anatomy 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 230000003834 intracellular effect Effects 0.000 abstract description 8
- 230000008859 change Effects 0.000 abstract description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 6
- 229910052760 oxygen Inorganic materials 0.000 abstract description 6
- 239000001301 oxygen Substances 0.000 abstract description 6
- 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
- 210000004027 cell Anatomy 0.000 description 68
- 239000000243 solution Substances 0.000 description 13
- 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
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000004691 chief cell of stomach Anatomy 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
- 238000002474 experimental method Methods 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
- 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
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses and a kind of utilize DAPI fluorescent dye to the detection method of active o content in living cells, 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 utilize DAPI fluorescent dye to the detection method of active o content in living cells, belong to biological
Competent 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 one and utilizes DAPI fluorescent dye to contain active oxygen in living cells
The detection method of amount, the method can obtain exposing under variable concentrations the DCF fluorescence signal of single living cell corresponding to pollutant
Value.
For solving above-mentioned technical problem, the technical solution adopted in the present invention is as follows:
A kind of utilize DAPI fluorescent dye to the detection method of active o content in living cells, comprise 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 DAPI Incubating Solutions to each orifice plate, at 37 DEG C, hatch 20min, after hatching, use PBS
Buffer solution, uses microplate reader to detect the DAPI fluorescence signal value of each orifice plate inner cell, obtains living cells in each orifice plate
Quantity；
Step 6, the DCF fluorescence signal value in each orifice plate step 4 obtained is divided by the DAPI 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.
Wherein, in step 1, first test cell strain PBS is cleaned；Use trypsin-EDTA molten after cleaning
Liquid peptic cell；Digestion is terminated by the culture medium containing serum after cell dissociation；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 DAPI Incubating Solution is 2.5 μ g/ml.
Wherein, in step 5, the excitation wavelength of the DAPI fluorescent dye that described DAPI Incubating Solution is corresponding is 350nm, transmitted wave
A length of 460nm.
Compared with existing detection method, the present invention utilizes DAPI fluorescent dye to the detection side of active o content in living cells
Method has the beneficial effect that
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 utilize DAPI fluorescent dye to the detection method of active o content in living cells, comprise 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 DCF Incubating Solution of final concentration of 10 μMs of 50 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 the DAPI Incubating Solution of the final concentration of 2.5 μ g/ml of 100 μ L, incubate at 37 DEG C
Educate 20min, wash 2 times with PBS after hatching, use microplate reader to detect the DAPI fluorescence signal value of each orifice plate inner cell,
Obtain 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 DAPI of cell in corresponding orifice plate
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；The excitation wave of DAPI fluorescent dye
A length of 350nm, a length of 460nm of transmitted wave.
As it is shown in figure 1, by DAPI fluorescence signal value it can be seen that along with expose pollutant arsenic (As) concentration increase,
In each orifice plate, viable count amount occurs that significance reduces；Fig. 2 is the DCF letter of each orifice plate after DAPI fluorescence signal value standardization
Number value, figure it is seen that be 5 μMs from exposing pollutant arsenic (As) concentration, the increase exposing pollutant arsenic (As) concentration is aobvious
Work 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 DCF Incubating Solution of final concentration of 10 μMs of 50 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 presenting very after DAPI fluorescence signal value standardized DCF fluorescence signal and standardization
Different: first, process group when exposure pollutant arsenic (As) concentration of significant difference is 5 μMs should be there is and do not show significantly
Sex differernce, its reason is that the minimizing of cell quantity makes total DCF fluorescence signal value change inconspicuous；Secondly, the experiment of Fig. 3
Result does not has obvious dose-effect relationship, and its reason have ignored cell quantity change equally and caused.
Claims (7)
1. one kind utilizes DAPI fluorescent dye to the detection method of active o content in living cells, it is characterised in that: include as follows
Step:
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 DAPI Incubating Solutions to each orifice plate, at 37 DEG C, hatch 20min, buffer with PBS after hatching
Liquid washs, and uses microplate reader to detect the DAPI fluorescence signal value of each orifice plate inner cell, 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 DAPI fluorescence of cell in corresponding orifice plate
Signal value, obtains the DCF fluorescence signal value of corresponding single living cell under different exposure pollutant levels.
The most according to claim 1 utilizing DAPI fluorescent dye to the detection method of active o content in living cells, it is special
Levy and be: in step 1, first test cell strain PBS is cleaned；Digest with trypsin-EDTA solutions after cleaning
Cell；Digestion is terminated by the culture medium containing serum after cell dissociation；Finally the cell suspension obtained is blown even.
The most according to claim 1 utilizing DAPI fluorescent dye to the detection method of active o content in living cells, it is special
Levying and be: in step 3, described exposure pollutant are arsenic.
The most according to claim 1 utilizing DAPI fluorescent dye to the detection method of active o content in living cells, it is special
Levying and be: in step 4, the concentration of described DCF Incubating Solution is 10 μMs.
The most according to claim 1 utilizing DAPI fluorescent dye to the detection method of active o content in living cells, it is special
Levying and be: in step 4, the excitation wavelength of the DCFH-DA fluorescent dye that described DCF Incubating Solution is corresponding is 485nm, and transmitted wave is a length of
530nm。
The most according to claim 1 utilizing DAPI fluorescent dye to the detection method of active o content in living cells, it is special
Levying and be: in step 5, the concentration of described DAPI Incubating Solution is 2.5 μ g/ml.
The most according to claim 1 utilizing DAPI fluorescent dye to the detection method of active o content in living cells, it is special
Levying and be: in step 5, the excitation wavelength of the DAPI fluorescent dye that described DAPI Incubating Solution is corresponding is 350nm, and transmitted wave is a length of
460nm。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610503947.3A CN106092991A (en) | 2016-06-29 | 2016-06-29 | A kind of utilize DAPI fluorescent dye to the detection method of active o content in living cells |
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| CN201610503947.3A CN106092991A (en) | 2016-06-29 | 2016-06-29 | A kind of utilize DAPI fluorescent dye to the detection method of active o content in living cells |
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Cited By (3)
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|---|---|---|---|---|
| CN109406471A (en) * | 2018-10-26 | 2019-03-01 | 安徽农业大学 | A kind of fluorescence imaging method with embryo's stereochemical structure that vaseline column is support |
| CN112326607A (en) * | 2020-10-16 | 2021-02-05 | 暨南大学 | Low-concentration ROS detection method and application thereof |
| CN112834473A (en) * | 2021-01-07 | 2021-05-25 | 华中科技大学 | Non-diagnosis-purpose quantitative detection method for active oxygen of single sperm and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406471A (en) * | 2018-10-26 | 2019-03-01 | 安徽农业大学 | A kind of fluorescence imaging method with embryo's stereochemical structure that vaseline column is support |
| CN112326607A (en) * | 2020-10-16 | 2021-02-05 | 暨南大学 | Low-concentration ROS detection method and application thereof |
| CN112834473A (en) * | 2021-01-07 | 2021-05-25 | 华中科技大学 | Non-diagnosis-purpose quantitative detection method for active oxygen of single sperm and application thereof |
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