CN106970051A - A kind of method that DNA Damage is caused based on high intension technology quantitative analysis tar - Google Patents
A kind of method that DNA Damage is caused based on high intension technology quantitative analysis tar Download PDFInfo
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Abstract
The invention discloses a kind of method that DNA Damage is caused based on high intension technology quantitative analysis tar, shown method comprises the following steps：1) cell culture, 2) cell contamination, 3) γ H2AX immunofluorescence label, 4) high intension technology quantitative analysis.The advantage of the invention is that：Using high intension imaging system automated imaging, the DNA double chain signs of failure thing γ H2AX protein that the induction of quantitative analysis tar is produced realizes direct, the quick detection of cell so that sample process is more convenient；High-resolution imaging, γ H2AX is directly observed in endonuclear distribution but also image document is easy to storage and is analyzed again, and it can realize that the γ H2AX induced in each nucleus tar carry out quantitative analysis, testing result is more sensitive and accurate.
Description
Technical field
The invention belongs to DNA damage vitro detection technical field, more particularly it relates to which tar causes cell DNA
The Quantitative in vitro analysis method of damage.
Background technology
Cigarette smoke can be divided into two-phase according to the state of material, and one kind is gas phase, and one kind is grain phase, and granule phase substance matter is Jiao
Oil.Contain a variety of noxious materials, such as superoxide anion O in coke tar in cigarette-, hydrogen peroxide isoreactivity oxygen class material, aldehydes, benzene
And pyrene etc..Tar can induce kinds cancer and breathing problem, and living radical and noxious material therein can be with inducing DNAs
Produce oxidative damage, DNA fracture, micronucleus, gene mutation etc..So, accurate detection tar to the damage of cell DNA for
The genetoxic and Evaluation of Harmfulness of tar have great importance.
DNA double chain fracture is a kind of damage type of DNA most serious, if this damage correctly or can not be repaiied in time
It may can cause chromosome aberration or Apoptosis etc. again.A kind of biomarker being broken as DNA double chain, phosphorylation
In terms of histone γ H2AX have been widely used in the researchs such as clinical medicine, radiology and toxicology.Including many single
Body compound and mixture are determined and evaluated to its genetoxic by γ H2AX experiments.For example, Tanaka etc. is utilized
γ H2AX, which are tested, to be detected and has been evaluated to the genetoxic of cigarette smoke condensate；Smart etc. is using γ H2AX experiments pair
The dose-effect relationship of the DNA double chain fracture of Etoposide, mitoxantrone and Methyl nitrosourea is studied.The experiment because
Its sensitivity is high, can carry out large scale analysis detection with reference to other instruments detection technique, possesses other genetoxics detection skill
A variety of advantages that art does not have, have been widely used in the genetoxic screening of compound and noxious material and have commented at present
Valency.
There are flow cytometer, ELISA (Enzyme-linked Immunosorbent Assay examinations to the γ H2AX main methods for analyze detection at present
Test), Western Blot (Western blotting), microscopy etc..Flow cytometer and Western Blot are cumbersome, inspection
Need attached cell digesting into single cell suspension before survey, destroy the 26S Proteasome Structure and Function integrality of cell, and detection flux compared with
It is low.ELISA can not provide the distribution situation of intracellular Fluorescence focus and need additionally to add other detection albumen to result progress
Correction, and the detection flux of microscopy is low, and the error artificially counted is larger.
The content of the invention
In view of the above-mentioned problems, it is an object of the invention to provide it is a kind of need not destroy cell, it is simple, effectively and sensitivity is high
Tar causes the quantitative analysis method of DNA damage, and the testing result of this method is accurate and visualizes, to overcome lacking for prior art
Fall into.
The purpose of the present invention is realized by the way that the quantitative analysis of high intension technology and γ H2AX is combined.Specifically,
The purpose of the present invention is achieved through the following technical solutions：
A kind of method that DNA Damage is caused based on high intension technology quantitative analysis tar, methods described includes following step
Suddenly：
1) cell culture：Cell is seeded in cell culture fluid and carries out cell culture；
2) cell is contaminated：The cell culture fluid is abandoned in suction, is added cell contamination liquid and is continued to cultivate, the cell contamination liquid bag
Containing tar and cell culture fluid；
3) immunofluorescence label：Cell contamination liquid is abandoned in suction, carry out in cell γ H2AX immunofluorescence label with it is thin
The DAPI dyeing of karyon；
4) high intension technology quantitative analysis：The fluoremetry of the nucleus and γ H2AX of the cell is carried out respectively, with institute
The average fluorescent strength that is detected in the effective cell core of the effective cell of identification characterizes γ H2AX content.
Preferably, the step 1) in, the cell is adherent growth cell；It is highly preferred that during inoculation, at the cell
In exponential phase, it is inoculated in single cell suspension in cell culture fluid；
It is highly preferred that cell inoculation after by cell in cell culture fluid in 37 DEG C, 5%CO2Under the conditions of cultivate 24h.
Preferably, the step 2) in, the tar is prepared by the following method：Standard cigarettes sample in temperature (22 ±
DEG C 1) and after 60% ± 3% time balance 48-72 of relative humidity, the cigarette sample is aspirated, then collection TPM adds DMSO
(dimethyl sulfoxide (DMSO)) is configured to save backup at the Smoke Particulate solution (tar) that concentration is 8-100mg/mL, -80 DEG C.According to
The embodiment of the present invention, the standard cigarettes sample is 3R4F.
Preferably, the concentration of tar is 0-500 μ g/mL in the cell contamination liquid, preferably>0 and≤500 μ g/mL, it is more excellent
Elect 3.91-500 μ g/mL as；
According to the embodiment of the present invention, in the cell contamination liquid concentration of tar can be 0,3.91,7.81,
15.63rd, 31.25,62.5,125,250 or 500 μ g/mL.
Preferably, the culture cell 1-24h, preferably 24h in cell contamination liquid.
Preferably, the step 3) include：
3-1) inhale and abandon the cell contamination liquid, wash cell, add paraformaldehyde progress cell and fix；
Cell 3-2) is washed, Triton-100X is then added so that cell-permeant；
Cell 3-3) is washed, seralbumin closing is then added, it is that anti-γ H2AX antibody is incubated that primary antibody is added afterwards
Educate；
Cell 3-4) is washed, the secondary antibody for then adding immunofluorescence label is incubated；
Cell 3-5) is washed, DAPI (4', 6- diamidino -2-phenylindone) is added and is dyed；
Cell 3-6) is washed, is preserved.
Preferably, the step 4) in, the nucleus of the cell is different at two groups respectively with γ H2AX fluoremetry
Excitation wavelength and launch wavelength under carry out；
Preferably, the step 3-4) in, the secondary antibody of immunofluorescence label is the secondary antibody that Alexa Fluor 488 are marked,
And the step 4) in, surveyed in passage one with the excitation wavelength 358nm and launch wavelength 461nm fluorescence for carrying out nucleus
It is fixed, γ H2AX fluoremetry is carried out with excitation wavelength 495nm and launch wavelength 519nm in passage two, to obtain passage one
The average fluorescent strength that the effective cell core internal channel two of the effective cell recognized is detected, thus characterizes containing for γ H2AX
Amount；
It is highly preferred that in two passages, measurement multiple is 200 times, 9 visuals field are analyzed and determined per hole.
According to the present invention embodiment, step 4) using high intension cell analysis system carry out, for example purchased from
Thermo Scientific model ArrayScan VTI600 high intension cell analysis system.
Optionally, method of the invention also includes, and is arranged on step 3-3) in without add primary antibody, only in step 3-4) in
Add secondary antibody blank control group so that in step 4) in obtain the blank signal as caused by non-specific adsorption, thus from detection
To fluoremetry signal in deduct blank signal.
The method of the present invention may also include the drafting of dose-effect curve.That is, by step 2) in set and a variety of include
The cell contamination liquid of the tar of various concentrations, carries out methods described, the average fluorescent strength and tar finally according to γ H2AX
Concentration draws dose-effect curve.
Or, the drafting of method of the invention m- effect curve when may also include.That is, by step 2) in by cell
Different time is cultivated in the cell contamination liquid of the tar comprising same concentrations, methods described is carried out, finally according to γ H2AX's
M- effect curve when average fluorescent strength and incubation time drafting.
According to the embodiment of the present invention, when cell behaviour lung cancer cell types, this hair can be carried out as follows
Bright method：
1) cell culture：Using the Glu containing 10%FBS and 2mmoL/L RPMI-1640 nutrient solutions in 37 DEG C,
5%CO2Under the conditions of in incubator cultivate human lung carcinoma cell line A549, when cell confluency rate reaches 70-80%, use
0.25% trypsase obtains single cell suspension after being digested, then using every μ L concentration of hole 100 as 105Individual cell/mL's
Inoculum concentration is inoculated in 96 porocyte culture plates, in the RPMI-1640 nutrient solutions of the Glu containing 10%FBS and 2mmoL/L
In 37 DEG C, 5%CO2Under the conditions of cultivate 24h.
2) cell is contaminated：The cell culture fluid abandoned after culture 24h is inhaled, cell contamination liquid is added and continues to cultivate, the cell
Contamination liquid is the step 1 for including 0,3.91,7.81,15.63,31.25,62.5,125,250 and 500 μ g/mL tar respectively) in
Cell culture fluid.Concentration group is only added containing 10%FBS and 2mmoL/L L- at least provided with two groups of blank controls, blank control group
The RPMI-1640 nutrient solutions of glutamine, in 37 DEG C, 5%CO2Under conditions of cultivate 24h.
3) immunofluorescence label：
3-1) inhale and abandon cell contamination liquid, 100 μ L PBS (phosphate buffers, pH 7.2~7.4 are added per hole；It is same in full)
Wash cell twice, every time at least 5min；Then the paraformaldehyde solution for 50 μ L 4% being added per hole is fixed at room temperature
15min；
Cell 3-2) fixed is washed twice with PBS again, every time at least 5min；Then add 50 μ L's 0.5%
Triton-100X solution (in PBS) penetrating 15min at room temperature；
Cell after 3-3) penetrating is washed twice with PBS again, every time at least 5min；Then 3%BSA confining liquids are added per hole
(in PBS) adds 50 μ L and contains mouse anti human γ H2AX antibody-solutions (1 afterwards in closing 1h at 37 DEG C:200, v/v), one
50 μ L 1%BSA are only added as negative control hole in group blank well, and another group of blank well adds 50 μ L and contain mouse anti human γ
H2AX antibody-solutions are as blank control wells, incubation conditions：Constant-temperature incubation 2h or 4 DEG C are overnight at 37 DEG C；
Cell after 3-4) primary antibody is incubated washs three times with PBS again, every time at least 5min；Then 50 μ L are added per hole
The goat anti-mouse antibody solution (1 that Alexa Fluro 488 are marked:200, v/v) it is incubated 2h in lucifuge at 37 DEG C；
Cell after 3-5) secondary antibody is incubated washs three times with PBS again, every time at least 5min；Then the μ g/mL of 50 μ L 1 are added
DAPI (in PBS) contaminate core 10min at room temperature；
After 3-6) PBS is washed three times, 100 μ L PBS are added per hole, 4 DEG C are kept in dark place, to be measured.
4) high intension technology quantitative analysis：Passage one (nucleus fluoremetry) sets excitation wavelength and launch wavelength difference
For 358nm and 461nm, passage two (object γ H2AX fluoremetry) setting excitation wavelength and launch wavelength are respectively
495nm and 519nm, measurement multiple is 200, is analyzed per hole and determines 9 visuals field, and the effective cell recognized with passage one has
The average fluorescent strength that effect nucleus internal channel two is detected characterizes γ H2AX content.
5) data processing：Be arranged on step 3-3) in without add primary antibody, only in step 3-4) in add secondary antibody blank
Control group, in step 4) in detect signal i.e. be the blank signal as caused by non-specific adsorption, from all samples instrument connection
Blank signal is deducted in detection signal；The concentration of fluorescence intensity and tar finally according to object γ H2AX draws dosage-effect
Answer curve.
Referring to Fig. 1, the invention provides a kind of side for causing DNA Damage come quantitative analysis tar based on high intension technology
Method, overcomes the deficiency of existing tar in-vitro contamination and DNA damage detection method.Specifically, commented the invention provides one kind
Estimate the method for DNA double chain crack conditions caused when contamination poisonous substance is tar, wherein phospho-histone γ H2AX are used as Jiao
The biomarker of the DNA double chain fracture of oil induction, and γ H2AX are detected using high intension technology, improve detection
Efficiency and sensitivity.In the method for the invention, cell passes through immunofluorescent staining γ H2AX and height after exposure tar
Intension automated imaging realizes the direct of cell sample, high flux with analysis and detected.
Compared with prior art, method of the invention also has following excellent results：
1) method of the invention can be carried out in Tissue Culture Plate, such as 96 orifice plates, therefore required cell concentration and sample
Amount is few, 96 samples can be detected simultaneously, detection flux is higher；
2) cell need not be destroyed before detecting, single cell suspension is prepared without enzymolysis or extracts albumen, so sample process
More easily and quickly；
3) there is high intension technology high-resolution imaging to obtain function, therefore γ H2AX can be straight in endonuclear distribution
Observation is connect, and image document also allows for storage in case analyzing again；
4) recognized by nucleus, quantitative analysis can be carried out to the γ H2AX of fluorescence labeling in each nucleus, so inspection
Survey result more sensitive and accurate.
Brief description of the drawings
Hereinafter, embodiment of the present invention is described in detail with reference to accompanying drawing, wherein：
Fig. 1 shows the flow chart of the inventive method.
Fig. 2 shows that tar induction A549 cells produce γ H2AX dose-effect curve, does not have in cell contamination liquid wherein
There is addition metabolism activation system rats'liver S9.
Fig. 3 shows that tar induction A549 cells produce γ H2AX dose-effect curve, wherein adds in cell contamination liquid
Plus metabolism activation system rats'liver S9.
Fig. 4 shows that tar induction A549 cells produce γ H2AX when m- effect curve.
Embodiment
Illustrate the present invention referring to specific embodiment.It will be appreciated by those skilled in the art that these embodiments are only
For illustrating the present invention, it does not limit the scope of the present invention in any way.
Experimental method in following embodiments, is conventional method unless otherwise specified.Medicine used in following embodiments
Product raw material, reagent material etc., unless otherwise specified, are commercially available products.
Primary antibody：Mouse anti human γ H2AX antibody, purchased from Biolegend, article No. 613402；
Solution is configured to when using：100 μ L mouse anti human γ H2AX antibody are taken to add in 1% BSA solution, 1:200(v/
V) dilute, fully mix.
Secondary antibody：The goat anti-mouse antibody that Alexa Fluro 488 are marked, purchased from Wuhan Jia Yuan quantum dots company, article No.
YM002；
Solution is configured to when using：It is molten that the goat anti-mouse antibody for taking 100 μ L Alexa Fluro 488 to mark adds PBS
In liquid, 1:200 (v/v) dilute, and fully mix.
Rats'liver S9 solution：Purchased from MolTox, article No. 11-101.1.
10%S9 mixtures are configured to when using：By 4 bulk solution A, 60 bulk solution B, 16 bulk solution C, 10 volumes
Solution D is mixed with 10 volume rats'liver S9 solution, and wherein solution A contains 0.2moL/L MgCl2With 0.825moL/L's
KCl；Solution B contains 0.176moL/L Na2H2PO4With 0.024moL/L NaH2PO4；Solution C contains 0.025moL/L's
NADP.Solution D contains 0.05moL/L G-6-P salt；Solution A-D is prepared using deionized water.
The preparation of tar：Standard cigarettes sample 3R4F is in 60% ± 3% time balance of temperature (22 ± 1) DEG C and relative humidity
After 48h, then with rotating disc type smoking machine smoking cigarette, TPM is collected using cambridge filter, a certain amount of DMSO is then added
(dimethyl sulfoxide (DMSO)) is configured to save backup at the Smoke Particulate solution (tar) that concentration is 100mg/mL, -80 DEG C.
High intension cell analysis system, purchased from Thermo Scientific, model ArrayScan VTI600.
Embodiment 1
When in cell contamination liquid without addition metabolism activation system rats'liver S9, determine what is induced after tar exposure 24h
γH2AX。
The A549 cells in exponential phase are collected, with every 10000 cell seedings in hole in 96 orifice plates, containing 10%
In 37 DEG C, 5%CO in the RPMI-1640 nutrient solutions of FBS and 2mmoL/L Glus2Incubator is incubated 24h.
Inhale abandon culture 24h after cell culture fluid, with respectively contain 0,3.91,7.81,15.63,31.25,62.5,125,
The cell culture fluid of 250 and 500 μ g/mL tar (is equally the RPMI-1640 of the Glu containing 10%FBS and 2mmoL/L
Nutrient solution) as cell contamination liquid, continue to cultivate cell 24h.
Contamination is inhaled after terminating abandons cell contamination liquid, and 100 μ L PBS are added per hole and are washed twice, every time at least 5min；Then
The paraformaldehyde solution that 50 μ L 4% are added per hole fixes 15min at room temperature；The cell fixed washs two with PBS
It is secondary, at least 5min every time；Then 50 μ L 0.5% Triton-100X solution penetrating 15min at room temperature is added per hole；After penetrating
Cells rinsed with PBS twice, at least 5min every time；Then 3%BSA confining liquids (in PBS) are added per hole in envelope at 37 DEG C
1h is closed, 50 μ L of each sample well addition contain mouse anti human γ H2AX antibody-solutions (1 afterwards:200, v/v), in one group of blank well
50 μ L 1%BSA are only added as negative control hole, it is molten that another group of 50 μ L of blank well addition contain mouse anti human γ H2AX antibody
Liquid is used as blank control wells.Incubation conditions are：Constant-temperature incubation 2h or 4 DEG C are overnight at 37 DEG C；Cell PBS after primary antibody incubation
Washing three times, every time at least 5min；Then the goat anti-mouse antibody that 50 μ L Alexa Fluro 488 marks are added per hole is molten
Liquid (1:200, v/v) it is incubated 2h in lucifuge at 37 DEG C；Cell after secondary antibody is incubated washs three times with PBS again, every time at least 5min；
Then lucifuge contaminates core 10min to every hole addition 50 μ L 1 μ g/mL DAPI (in PBS) at room temperature；After PBS is washed three times, often
Hole adds 100 μ L PBS, and 4 DEG C are kept in dark place.
It is automatic focus on after, set the passage one (nucleus fluoremetry) of high intension cell analysis system excitation wavelength and
Launch wavelength is respectively 358nm and 461nm, sets excitation wavelength and the transmitting of passage two (object γ H2AX fluoremetry)
Wavelength is respectively 495nm and 519nm, and measurement multiple is 200 times, 9 visuals field is analyzed and determined per hole, γ H2AX are with the institute of passage one
The average fluorescent strength that the effective cell core internal channel two of the effective cell of identification is determined is characterized.
Blank control group is set in experiment, i.e., does not add primary antibody, only adds two antiantibodys of fluorescence labeling, detect letter
Number i.e. be the blank signal as caused by non-specific adsorption；The detection signal of all samples instrument connection should deduct blank signal；Finally
Dose-effect curve is drawn according to object γ H2AX fluorescence intensity and the concentration of tar.
Fig. 2 show the tar of various concentrations γ H2AX that induction A549 cells are produced after contamination 24h docs-effect
Relation curve.As seen from the figure, with the rise of tar-concentration, the γ H2AX that A549 cells are produced gradually rise, with obvious
Dose-effect relationship.When tar concentration of contamination is 250 μ g/mL, the γ H2AX of intracellular generation exceed normal group (i.e. tar
Concentration be 0 when) 1.5 times.
Embodiment 2
When adding metabolism activation system rats'liver S9 in cell contamination liquid, the γ induced after tar exposure 24h is determined
H2AX。
Experimentation according to described in embodiment 1 carry out, only difference is that, cell contamination liquid remove comprising cell culture fluid with
Outside tar, mixed with 10%S9 mixtures so that contain 1%S9 mixtures in cell contamination liquid.
Fig. 3 is shown in cell contamination liquid after addition 1%S9, and the tar of various concentrations induces A549 thin after contamination 24h
The dose-effect relationship curve for the γ H2AX that born of the same parents produce.As figure shows, with the increase of concentration of contamination, the γ of intracellular generation
H2AX constantly gradually rises, and shows significant dose-effect relationship.When the concentration of tar is 250 μ g/mL, intracellular production
Raw γ H2AX exceed 1.5 times of normal group.
Addition S9 can strengthen the metabolic conversion of tar in vitro, therefore addition hydra assay system in cell contamination liquid
System S9 can avoid in testing in vitro due to cellular metabolic activity it is not enough caused by test result be false negative possibility.Cause
This, can more accurately understand induction of the tar in vitro to DNA Damage, and thus prove by addition and without S9
The method of the present invention is objective, available.
Embodiment 3
When in cell contamination liquid without addition metabolism activation system rats'liver S9, determine 250 μ g/mL tar and expose respectively
1st, the γ H2AX induced after 2,4,8,12 and 24h.
Experimentation is carried out according to described in embodiment 1, only difference is that, concentration of the tar in cell contaminates liquid is fixed
In the μ g/mL of concentration 250, contamination time is respectively 1,2,4,8,12 and 24h.
Fig. 4 show the 250 μ g/mL tar γ that induction A549 cells are produced after 1,2,4,8, the 12 and 24h that contaminates
H2AX when m- effect relation curve.As seen from the figure, as contamination time increases, the γ H2AX that A549 cells are produced gradually increase
It is many, m- effect relation when showing significant.
Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can be according to this
Invention is variously modified or deformed, and without departing from the spirit of the present invention, all should belong to the model of appended claims of the present invention
Enclose.
Claims (9)
1. a kind of method that DNA Damage is caused based on high intension technology quantitative analysis tar, the described method comprises the following steps：
1) cell culture：Cell is seeded in cell culture fluid and carries out cell culture；
2) cell is contaminated：The cell culture fluid is abandoned in suction, is added cell contamination liquid and is continued to cultivate, the cell contamination liquid includes Jiao
Oil and cell culture fluid；
3) immunofluorescence label：The cell contamination liquid is abandoned in suction, carries out the immunofluorescence label and nucleus of γ H2AX in cell
DAPI dyeing；
4) high intension technology quantitative analysis：The fluoremetry of the nucleus and γ H2AX of the cell is carried out respectively, to be recognized
Effective cell effective cell core in the average fluorescent strength that detects characterize γ H2AX content.
2. according to the method described in claim 1, it is characterised in that the step 1), the cell is adherent growth cell；
Preferably, during inoculation, the cell is in exponential phase, is inoculated in single cell suspension in cell culture fluid；
It is highly preferred that cell inoculation after by cell in cell culture fluid in 37 DEG C, 5%CO2Under the conditions of cultivate 24h.
3. method according to claim 1 or 2, it is characterised in that the step 2) in, the tar is by the following method
Prepare：Standard cigarettes sample aspirates the cigarette in after 60% ± 3% time balance 48-72h of temperature (22 ± 1) DEG C and relative humidity
Sample, collects TPM, then adds DMSO (dimethyl sulfoxide (DMSO)) and is configured to the Smoke Particulate that concentration is 8-100mg/mL
Solution (tar), is saved backup at -80 DEG C.
4. according to the method in any one of claims 1 to 3, it is characterised in that the step 2), the cell contamination
The concentration of tar is 0-500 μ g/mL in liquid, preferably>0 and≤500 μ g/mL, more preferably 3.91-500 μ g/mL；
Preferably, the culture cell 1-24h, preferably 24h in cell contamination liquid.
5. method according to any one of claim 1 to 4, it is characterised in that the step 3) include：
3-1) inhale and abandon the cell contamination liquid, wash cell, add paraformaldehyde progress cell and fix；
Cell 3-2) is washed, Triton-100X is then added so that cell-permeant；
Cell 3-3) is washed, seralbumin closing is then added, it is that anti-γ H2AX antibody is incubated that primary antibody is added afterwards；
Cell 3-4) is washed, the secondary antibody for then adding immunofluorescence label is incubated；
Cell 3-5) is washed, DAPI is added and is dyed；
Cell 3-6) is washed, is preserved.
6. method according to any one of claim 1 to 5, it is characterised in that the step 4) in, the cell it is thin
Karyon and γ H2AX fluoremetry are carried out under two groups of different excitation wavelengths and launch wavelength respectively；
Preferably, the step 3-4) in, the secondary antibody of immunofluorescence label is the secondary antibody that Alexa Fluor 488 are marked, and
The step 4) in, the fluoremetry of nucleus is carried out with excitation wavelength 358nm and launch wavelength 461nm in passage one,
γ H2AX fluoremetry is carried out in passage two with excitation wavelength 495nm and launch wavelength 519nm, is recognized with obtaining passage one
Effective cell the average fluorescent strength that is detected of effective cell core internal channel two, thus characterize γ H2AX content；
Preferably, in two passages, measurement multiple is 200 times, and 9 visuals field are analyzed and determined per hole.
7. method according to any one of claim 1 to 6, it is characterised in that optionally, methods described also includes, if
Put in step 3-3) in without add primary antibody, only in step 3-4) in add secondary antibody blank control group so that in step 4) in
The blank signal as caused by non-specific adsorption is obtained, thus blank signal is deducted from the fluoremetry signal detected.
8. method according to any one of claim 1 to 7, it is characterised in that by step 2) the middle a variety of bags of setting
Tar containing various concentrations cell contamination liquid, carry out methods described, finally according to step 4) obtain γ H2AX mean fluorecences
Intensity and the concentration of tar draw dose-effect curve.
9. method according to any one of claim 1 to 7, it is characterised in that by step 2) in by cell in bag
Different time is cultivated in the cell contamination liquid of tar containing same concentrations, methods described is carried out, finally according to step 4) obtain
M- effect curve when γ H2AX average fluorescent strengths and incubation time drafting.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107764716A (en) * | 2017-10-21 | 2018-03-06 | 云南中烟工业有限责任公司 | A kind of detection method of cigarette smoke to cellular water Permeability |
| WO2019208920A1 (en) * | 2018-04-27 | 2019-10-31 | 한국화학연구원 | Composition for immunostaining cleared large tissues and method for immunostaining cleared large biological tissues |
| CN111521587A (en) * | 2020-04-24 | 2020-08-11 | 中国烟草总公司四川省公司 | Method for detecting phosphorylation level of tyrosine in cell |
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| CN107764716B (en) * | 2017-10-21 | 2020-08-14 | 云南中烟工业有限责任公司 | Method for detecting influence of cigarette smoke on cell water permeability |
| WO2019208920A1 (en) * | 2018-04-27 | 2019-10-31 | 한국화학연구원 | Composition for immunostaining cleared large tissues and method for immunostaining cleared large biological tissues |
| KR20190124902A (en) * | 2018-04-27 | 2019-11-06 | 한국화학연구원 | Composition for immunostaining of cleared huge biological tissue and immunostaining method for cleared huge tissue |
| KR102141496B1 (en) * | 2018-04-27 | 2020-08-06 | 한국화학연구원 | Composition for immunostaining of cleared huge biological tissue and immunostaining method for cleared huge tissue |
| US11391653B2 (en) | 2018-04-27 | 2022-07-19 | Korea Research Institute Of Chemical Technology | Composition for immunostaining cleared large tissues and method for immunostaining cleared large biological tissues |
| CN111521587A (en) * | 2020-04-24 | 2020-08-11 | 中国烟草总公司四川省公司 | Method for detecting phosphorylation level of tyrosine in cell |
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