CN107064481B - A kind of liquid-vapor interface exposure system combines the method for high intension technology quantitative detection carbon monoxide cause DNA Damage - Google Patents
A kind of liquid-vapor interface exposure system combines the method for high intension technology quantitative detection carbon monoxide cause DNA Damage Download PDFInfo
- Publication number
- CN107064481B CN107064481B CN201710156524.3A CN201710156524A CN107064481B CN 107064481 B CN107064481 B CN 107064481B CN 201710156524 A CN201710156524 A CN 201710156524A CN 107064481 B CN107064481 B CN 107064481B
- Authority
- CN
- China
- Prior art keywords
- cell
- carbon monoxide
- h2ax
- added
- contamination
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
Abstract
The invention discloses a kind of liquid-vapor interface exposure systems to combine the method that high intension technology quantitative detection carbon monoxide causes DNA Damage, shown method is the following steps are included: 1) cell culture, 2) it is contaminated by the carbon monoxide of liquid-vapor interface exposure chamber, 3) immunofluorescence label of γ H2AX, 4) high intension detection.The present invention has the advantages that realizing gaseous carbon monoxide in vitro to the efficient contamination of attached cell using external liquid-vapor interface exposure chamber, the contamination efficiency of carbon monoxide is improved；Using high intension imaging system automated imaging, the DNA double chain signs of failure object γ H2AX protein that the induction of quantitative analysis carbon monoxide generates realizes direct, the quick detection of cell, so that sample process is more convenient；High-resolution imaging, it observe that γ H2AX can directly in endonuclear distribution but also analyze image document convenient for storage and again, and the γ H2AX induced in each nucleus carbon monoxide may be implemented and carry out quantitative analysis, testing result is more sensitive and accurate.
Description
Technical field
The invention belongs to external genetoxic determination techniques fields, more particularly it relates to the something lost of carbon monoxide
Pass toxicity test method.
Background technique
Carbon monoxide in air is mainly to be generated by organic matter imperfect combustion, and vehicle exhaust, coal burning etc. are
Its main source.Carbon monoxide can compete jointly with oxygen and the combination of hemoglobin, leads to histanoxia to generate poison
Property.But the genetoxic about carbon monoxide rarely has research at present, animal experiments show that carbon monoxide exposure rat urine without
Mutability.But carbon monoxide may participate in oxidative stress and DNA damage repair process.World Health Organization tobacco control at present
Carbon monoxide is classified as one of flue gas priority control pollutant by framework convention (FCTC) processed.Carbon monoxide genetoxic research compared with
Few reason mainly has its neurotoxicity and acute toxicity larger, therefore genetoxic suffers from ignorance；And carbon monoxide
Exposed technology is immature in vitro, it is difficult to effectively be studied.
As a kind of biomarker of DNA double chain fracture, phospho-histone γ H2AX has been widely used in
The research aspect such as clinical medicine, radiology and toxicology.It is tested including many monomeric compounds and mixture by γ H2AX
Its genetoxic is determined and is evaluated.For example, Tanaka etc. tests the something lost to cigarette smoke condensate using γ H2AX
Toxicity is passed to be detected and evaluated；Smart etc. is tested using γ H2AX to Etoposide, mitoxantrone and Methyl nitrosourea
The dose-effect relationship of DNA double chain fracture is studied, and by known different type genetoxic compound to this
Experiment is evaluated.The experiment can carry out large scale analysis detection because of its sensitivity height, in conjunction with other instruments detection technique,
Possess a variety of advantages that other genetic toxicology assays do not have, is widely used in compound and toxic at present
The genetoxic of substance screens and evaluation.
Having flow cytometer, ELISA to the γ H2AX main method for carrying out analysis detection at present, (Enzyme-linked Immunosorbent Assay tries
Test), Western Blot (Western blotting), microscopy etc..Flow cytometer and Western Blot are cumbersome, inspection
Need to digest attached cell at single cell suspension before survey, destroy the structure and function integrality of cell, and detect flux compared with
It is low.ELISA cannot provide the distribution situation of intracellular Fluorescence focus and need additionally to add other detection albumen and carry out to result
Correction, and the detection flux of microscopy is low, and the error artificially counted is larger.
Due to the limitation of technology and condition, traditionally the exposure for gaseous state substance and toxicity detection are usually by gaseous state object
Matter is dissolved in liquid, to realize contact exposure of the cell with gas, i.e. gas-liquid mixing exposure chamber.This exposure chamber
It is not often effective although useful for many substances.This is because some gases are dissolved in liquid (usually water)
Solubility it is limited, to limit the upper limit of exposure concentrations；Second, some gaseous materials can react with water, thus
It connects and changes testing goal；Third, although gas-liquid mixing exposure can make to be dissolved in fluid matrix for suspension cell
Gas comes into full contact with cell, but the cell of adhere-wall culture is difficult to realize and is come into full contact with.
The development of liquid-vapor interface exposure system largely avoid traditional gas-liquid mixing exposure chamber there are the problem of,
Greatly improve the validity that attached cell exposes in vitro.Therefore, liquid-vapor interface system has been widely used for studying at present
The exposure of volatile materials or aerosol substance.
Summary of the invention
In view of the above-mentioned problems, the object of the present invention is to provide a kind of without destroying cell, simple, effectively and high sensitivity
The quantitative detecting method of carbon monoxide DNA damage, this method can be carried out using direct gas exposure chamber, and testing result is quasi-
It really and visualizes, to overcome the deficiencies of existing technologies, to provide effective detection means of carbon monoxide genetoxic.
The purpose of the present invention is real and γ H2AX technology combines by detecting liquid-vapor interface exposure technology and high intension
It is existing.Specifically, the purpose of the present invention is achieved through the following technical solutions:
A kind of liquid-vapor interface exposure system combines the side of high intension technology quantitative detection carbon monoxide cause DNA Damage
Method the described method comprises the following steps:
1) cell culture: by cell inoculation in the device that bottom is permeable membrane, then by described device in cell
Cell culture is carried out in culture solution, makes cell monolayer growth on permeable membrane；
2) cell contaminate: remove described device in cell culture fluid, withdrawing device be placed in comprising cell contamination liquid and
In the contamination storehouse of poisonous gas, so that the permeable membrane bottom in device is contacted with cell contamination liquid, while permeable membrane
On cell surface be exposed to poisonous gas, wherein the poisonous gas include carbon monoxide and air；
3) immunofluorescence label: described device is taken out from contamination storehouse, carries out the immunofluorescence label of γ H2AX in cell
It is dyed with the DAPI of nucleus；
4) high intension technology quantitative detection: the fluoremetry of the nucleus and γ H2AX of the cell is carried out, respectively with institute
The average fluorescent strength detected in the effective cell core of the effective cell of identification characterizes the content of γ H2AX.
Preferably, in the step 1), the cell is adherent growth cell；It is highly preferred that when inoculation, at the cell
In logarithmic growth phase, it is inoculated in described device with single cell suspension；
It is highly preferred that the cell is inoculated in bottom as in the device of permeable membrane, then thin using single cell suspension
In 37 DEG C in born of the same parents' culture solution, 5%CO2Under the conditions of cultivate for 24 hours；
Preferably, about the bottom of the invention used for the device of permeable membrane, the permeable membrane is dacron membrane,
The aperture of aperture is 0.4 μm on film.Therefore, when described device to be carried out to cell culture in cell culture fluid, cell culture fluid
The cell of inoculation thereon can be submerged by permeable membrane；
Preferably, before cell inoculation, described device is balanced into 1-2h at 37 DEG C with cell culture fluid or PBS.
Preferably, in the step 2), the cell contamination liquid in the contamination storehouse includes the cell culture fluid in step 1),
It is preferred that identical as the cell culture fluid in step 1)；
Preferably, the poisonous gas is made of air and carbon monoxide；
It is highly preferred that in the poisonous gas carbon monoxide concentration be 0-44.64mmoL/L, preferably > 0 and≤
44.64mmoL/L more preferable 8.93-44.64mmoL/L；
Specific embodiment according to the present invention, in the poisonous gas concentration of carbon monoxide can be 0,8.93,
17.86,26.78,35.71 or 44.64mmoL/L.
Preferably, the flow of the poisonous gas is 10-50mL/min, and preferably 20mL/min preferably connects with poisonous gas
Touching≤2h, preferably 1h.Containing cell contamination liquid in contamination storehouse, but the cellular portions on permeable membrane are exposed to cell contamination
On the gas-liquid interface of liquid, to realize that the expose portion is contacted with the poisonous gas in storehouse of contaminating.
Preferably, the step 3) includes:
Described device 3-1) is taken out from contamination storehouse, washs, is subsequently placed in pallet, is added in the cell into device more
Polyformaldehyde carries out cell and fixes；
Described device and pallet 3-2) are washed, TritonX-100 then is added in the cell into device so that cell is logical
Thoroughly；
Described device and pallet 3-3) are washed, seralbumin closing, Zhi Houjia are then added in the cell into device
Enter the i.e. anti-γ H2AX antibody of primary antibody to be incubated for；
Described device and pallet 3-4) are washed, the secondary antibody that immunofluorescence label is then added in the cell into device carries out
It is incubated for；
Described device and pallet 3-5) are washed, DAPI (4', 6- diamidino -2- benzene is then added in the cell into device
Base indoles) it is dyed；
Described device and pallet 3-6) are washed, is saved.
Preferably, in the step 4), the nucleus of the cell and the fluoremetry of γ H2AX are respectively in two groups of differences
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 is marked,
And in the step 4), surveyed in channel one with the fluorescence that excitation wavelength 358nm and launch wavelength 461nm carries out nucleus
It is fixed, the fluoremetry of γ H2AX is carried out, with excitation wavelength 495nm and launch wavelength 519nm in channel two to obtain channel one
Thus the average fluorescent strength detected of effective cell core internal channel two of the effective cell identified characterizes containing for γ H2AX
Amount；
It is highly preferred that measurement multiple is 100 times in two channels, every hole analysis and 9 visuals field of measurement.
Specific embodiment according to the present invention, step 4) are carried out using high intension cell analysis system, such as purchased from
The high intension cell analysis system of the model ArrayScan VTI600 of Thermo Scientific.
Optionally, method of the invention further includes being arranged in step 3-3) that primary antibody is not added, only in step 3-4)
The blank control group of secondary antibody is added, so that the blank signal as caused by non-specific adsorption is obtained in step 4), thus from detection
To fluoremetry signal in deduct blank signal.
Method of the invention may also include the drafting of dose-effect curve.That is, including by the way that setting is a variety of in step 2)
The poisonous gas of the carbon monoxide of various concentration carries out the method, finally according to the average fluorescent strength of γ H2AX and an oxygen
The concentration for changing carbon draws dose-effect curve.
Alternatively, the drafting of method of the invention m- effect curve when may also include.That is, by making cell in step 2)
The poisonous gas of carbon monoxide of the contact comprising same concentrations reaches different time in contamination storehouse, carries out the method, last root
M- effect curve when being drawn according to the average fluorescent strength of γ H2AX and the time of contact toxicity gas.
Preferably, the poisonous gas enters in the contamination storehouse after 0.2 μm of membrane filtration.It is described in discharge
Poisonous gas can be still discharged in exhaust pipe after 0.2 μm of membrane filtration.
Specific embodiment according to the present invention, the device of permeable membrane is in the bottom used in the method for the invention
It is commercially available Transwell plug-in unit, such as the Transwell plug-in unit of the model 3450 purchased from Corning, device with pallet
At the top of Transwell plug-in unit, pallet is Transwell plug-in unit bottom.When cell is people's lung cancer cell types, Ke Yiru
It is lower to carry out method of the invention:
1) cell culture: using the L-Glutamine containing 10%FBS and 2mmoL/L RPMI-1640 culture solution in 37 DEG C,
5%CO2Under the conditions of cultivate human lung carcinoma cell line A549 in the incubator, when cell confluency rate reaches 70-80%, use
0.25% trypsase obtains single cell suspension after being digested；
Before inoculation, PBS (phosphate buffer, the pH of 0.5 and 2mL are separately added at the top and bottom of Transwell plug-in unit
7.2~7.4；Full text is same), 1-2h is balanced under conditions of 37 DEG C；The PBS abandoned at the top and bottom of Transwell plug-in unit is inhaled, in slotting
It is 4 × 10 that 0.5mL concentration is added at the top of part5The single cell suspension of a cell/mL is added 2mL in plug-in unit bottom and contains
The RPMI-1640 culture solution of 0.01moL/L HEPES, 2mmoL/L L-Glutamine and 10%FBS；Bottom will be put at the top of plug-in unit
In portion, and in 37 DEG C, 5%CO2Under the conditions of cultivate for 24 hours.
2) cell is contaminated: being removed the culture solution at the top of Transwell plug-in unit, is taken out and be put into body at the top of Transwell plug-in unit
In the contamination storehouse of outer exposing device, so that the permeable membrane in Transwell plug-in unit is contacted with cell contamination liquid, permeability filter
Cell surface on film is exposed to the poisonous gas 1h in contamination storehouse；The poisonous gas divides carbon monoxide by using air
It is not diluted to the concentration of 0,8.93,17.86,26.78,35.71 and 44.64mmoL/L, flow 20mL/min.
3) immunofluorescence label:
After 3-1) contaminating, taken out at the top of Transwell plug-in unit from contamination storehouse, then at the top of Transwell plug-in unit
It is separately added into 1mL PBS with bottom to wash twice, every time at least 5min；Then it will be put into bottom at the top of Transwell plug-in unit,
The paraformaldehyde solution that 0.5mL 4% is added into top fixes 15min at room temperature；
It 3-2) is separately added into 1mL PBS at the top and bottom of Transwell plug-in unit to wash twice, every time at least 5min, so
The TritonX-100 solution (in PBS) of 0.5mL 0.5% is added at the top of backward Transwell plug-in unit, it is penetrating at room temperature
15min；
It 3-3) is separately added into 1mL PBS at the top and bottom of Transwell plug-in unit to wash twice, every time at least 5min, so
The 3%BSA solution (in PBS) of 0.5mL is added at the top of backward Transwell plug-in unit, 1h is closed at 37 DEG C, then to
Addition 0.5mL contains mouse anti human γ H2AX antibody-solutions (1:200, v/v) constant temperature at 37 DEG C and incubates at the top of Transwell plug-in unit
Educate 2h；
1mL PBS washing 3-4) is separately added at the top and bottom of Transwell plug-in unit three times, every time at least 5min, so
Be added at the top of backward Transwell plug-in unit goat anti-mouse antibody solution that 0.5mL Alexa Fluor 488 marks (1:200,
V/v it) is protected from light at 37 DEG C and is incubated for 2h；
1mL PBS washing 3-5) is separately added at the top and bottom of Transwell plug-in unit three times, every time at least 5min, so
The DAPI solution (in PBS) that 500 μ L, 1 μ g/mL is added at the top of backward Transwell plug-in unit is protected from light dye core at room temperature
10min；
After 3-6) being separately added into 2mL PBS washing three times at the top and bottom of Transwell plug-in unit, then it is separately added into 0.5
With 2mL PBS, 4 DEG C are kept in dark place, to be measured.
4) high intension technology quantitative detection: excitation wavelength and launch wavelength difference is arranged in channel one (nucleus fluoremetry)
For 358nm and 461nm, channel two (fluoremetry of object γ H2AX) setting excitation wavelength and launch wavelength are respectively
495nm and 519nm, measurement multiple are 100, and every hole analysis and 9 visuals field of measurement, the effective cell identified with channel one have
Imitate the content of the average fluorescent strength detected of nucleus internal channel two characterization γ H2AX.
5) data processing: it is arranged in step 3-3) that primary antibody is not added, only in the middle blank that secondary antibody is added of step 3-4)
Control group, detected signal is the blank signal as caused by non-specific adsorption in step 4), from all samples instrument connection
Blank signal is deducted in detection signal；Agent is finally drawn according to the fluorescence intensity of object γ H2AX and the concentration of carbon monoxide
Amount-effect curve.
Referring to Fig. 1, the present invention provides one kind based on liquid-vapor interface exposure system and high intension technology come quantitative detection one
The method that carbonoxide causes DNA Damage, overcomes existing CO gas in-vitro contamination and DNA damage detection method not
Foot.Specifically, the present invention provides DNA double chains caused when a kind of assessment contamination poisonous substance is CO gas to be broken feelings
The method of condition, the biomarker for the DNA double chain fracture that wherein phospho-histone γ H2AX is induced as carbon monoxide, and
The efficiently and accurately control that gaseous carbon monoxide makes cells in vitro contaminate is realized using external liquid-vapor interface exposure chamber, is adopted
γ H2AX is detected with high intension technology, improves detection efficiency and sensitivity.In the method for the invention, it is grown on infiltration
Attached cell on permeability filter membrane after exposure carbon monoxide, passes through immunofluorescent staining in liquid-vapor interface exposure system
γ H2AX and high intension automated imaging and analysis realize direct, the high-throughput detection of cell sample.
Method of the invention has following technical characterstic:
Firstly, method of the invention has determined exponential phase cell monolayer growth on permeable membrane, thus with toxicity
During the contamination of gas, it is ensured that gas can come into full contact with cell, especially with respect to traditional gas-liquid mixed contamination side
For formula, the contamination efficiency of attached cell is significantly improved；
Second, to reduce influence of the gas impact to cell activity, the present invention is to different gas flow to cell activity
Influence is optimized.The study found that the impact of different gas flow 10-50mL/min formation is to the survival rate of cell without bright
Aobvious influence (p > 0.05), as shown in Figure 2, it is ensured that can effectively survive in this range of flow inner cell, and then guarantee contamination
Effect.Influence in conjunction with exposure warehouse product and contamination gas to environment, present invention preferably employs the gas flow of 20mL/min works
For final contamination flow.Also, also the use concentration of carbon monoxide is optimized with conditions such as contamination times by the present invention.
Compared with prior art, method of the invention also has following excellent results:
1) attached cell is which thereby enhanced instead of traditional gas-liquid mixing exposure chamber with liquid-vapor interface exposure chamber
With the contacting efficiency of carbon monoxide, and then the validity and accuracy of contamination are improved；
2) without destroying cell before detection, single cell suspension is prepared without enzymatic hydrolysis or extracts albumen, so sample process
More easily and quickly；
3) high intension technology has the function of that high-resolution imaging obtains, thus γ H2AX endonuclear distribution can be straight
Observation is connect, and image document is also convenient for storage in case analyzing again；
4) it is identified by nucleus, it can be to the γ H2AX progress quantitative analysis of fluorescent marker in each nucleus, so inspection
It is more sensitive and accurate to survey result.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:
Fig. 1 shows the flow chart of the method for the present invention.
Influence Fig. 2 shows different gas flow to cell survival rate.
Fig. 3 shows the dose-effect curve that carbon monoxide induction A549 cell generates γ H2AX.
Fig. 4 shows the when m- effect curve that carbon monoxide induction A549 cell generates γ H2AX.
Specific embodiment
The present invention is described below with reference to specific embodiments.It will be appreciated by those skilled in the art that these embodiments are only
For illustrating the present invention, do not limit the scope of the invention in any way.
Experimental method in following embodiments is unless otherwise specified conventional method.Medicine as used in the following examples
Product raw material, reagent material etc. are commercially available products unless otherwise specified.
Primary antibody: mouse anti human γ H2AX antibody is purchased from Biolegend, article No. 613402；
It is configured to solution when use: taking 100 μ L mouse anti human γ H2AX antibody to be added in 1% BSA solution, 1:200 (v/
V) it dilutes, mixes well.
Secondary antibody: the goat anti-mouse antibody that Alexa Fluro 488 is marked is purchased from Wuhan Jia Yuan quantum dot company, article No.
YM002；
Solution is configured to when use: it is molten that PBS is added in the goat anti-mouse antibody for taking 100 μ L Alexa Fluro 488 to mark
In liquid, 1:200 (v/v) dilution is mixed well.
Transwell plug-in unit: Corning, model 3450 are purchased from.
High intension cell analysis system is purchased from Thermo Scientific, model ArrayScan VTI600.
Cell exposure system, purchased from the intelligent honor in Beijing and model HRH-CES3969.
Embodiment 1
The γ H2AX induced after measurement carbon monoxide exposure 1h.
It is separately added into the PBS of 0.5 and 2mL at the top and bottom of Transwell plug-in unit, balances 1- under conditions of 37 DEG C
2h.The PBS abandoned at the top and bottom of Transwell plug-in unit is inhaled, it is 4 × 10 that 0.5mL concentration is added at the top of plug-in unit5A cell/mL
The single cell suspension of the A549 cell of logarithmic growth phase is added 2mL in plug-in unit bottom and contains 0.01moL/L HEPES, 2mmoL/L
The RPMI-1640 culture solution of L-Glutamine and 10%FBS, and in 37 DEG C, 5%CO2Under the conditions of cultivate for 24 hours.
The culture solution at the top of Transwell plug-in unit is removed, takes out and is put into cell exposure system at the top of Transwell plug-in unit
It contaminates in storehouse, so that the permeable membrane and cell contamination liquid in Transwell plug-in unit (are similarly containing 0.01moL/L
The RPMI-1640 culture solution of HEPES, 2mmoL/L L-Glutamine and 10%FBS) it contacts, the cell table on permeable membrane
Face is exposed to the poisonous gas in contamination storehouse, and wherein poisonous gas is made of carbon monoxide and air, the concentration point of carbon monoxide
Not Wei 0,8.93,17.86,26.78,35.71 and 44.64mmoL/L, contaminate 1h under conditions of 37 DEG C, and gas flow is
20mL/min。
After contamination, takes out at the top of Transwell plug-in unit from contamination storehouse, divide at the top and bottom of Transwell plug-in unit
Not Jia Ru 1mL PBS wash twice, at least 5min every time；Then it will be put into bottom at the top of Transwell plug-in unit, top is added
The paraformaldehyde solution of 0.5mL 4% fixes 15min at room temperature；1mL is separately added at the top and bottom of Transwell plug-in unit
PBS is washed twice, every time at least 5min, then to the TritonX-100 of addition 0.5mL 0.5% at the top of Transwell plug-in unit
Solution (in PBS), at room temperature penetrating 15min；1mL PBS washing is separately added at the top and bottom of Transwell plug-in unit
Twice, at least 5min every time, then to the 3%BSA solution (in PBS) that 0.5mL is added at the top of Transwell plug-in unit, in 37
1h is closed at DEG C, then to be added at the top of Transwell plug-in unit 0.5mL contain mouse anti human γ H2AX antibody-solutions (1:200,
V/v) the constant-temperature incubation 2h at 37 DEG C；1mL PBS washing is separately added at the top and bottom of Transwell plug-in unit three times, every time
At least 5min, then to the goat anti-mouse antibody that addition 0.5mL Alexa Fluor 488 is marked at the top of Transwell plug-in unit
Solution (1:200, v/v) is protected from light at 37 DEG C is incubated for 2h；1mL PBS is separately added at the top and bottom of Transwell plug-in unit to wash
It washs three times, every time at least 5min, then to the DAPI solution of addition 1 μ g/mL of 0.5mL at the top of Transwell plug-in unit (in PBS
In) it is protected from light dye core 10min at room temperature；After being separately added into 2mL PBS washing three times at the top and bottom of Transwell plug-in unit,
It is separately added into 0.5 and 2mL PBS in top and bottom, 4 DEG C are kept in dark place, to be measured.
The excitation wavelength and launch wavelength point in the channel one (nucleus fluoremetry) of high intension cell analysis system are set
Not Wei 358nm and 461nm, be arranged channel two (fluoremetry of object γ H2AX) excitation wavelength and launch wavelength be respectively
495nm and 519nm, measurement multiple are 100 times, and every hole analysis and 9 visuals field of measurement, the content of γ H2AX are identified with channel one
The average fluorescent strength detected of effective cell core internal channel two of effective cell characterized.
Blank control group is set in test, i.e., that primary antibody is not added, two antiantibodys of fluorescent marker is only added, 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 the fluorescence intensity of object γ H2AX and the concentration of carbon monoxide.
The dosage-for the γ H2AX that the carbon monoxide that Fig. 3 show various concentration induces A549 cell to generate after the 1h that contaminates
Effect relation curve.As seen from the figure, as the concentration of carbon monoxide increases, the γ H2AX that A549 cell generates is (i.e. than normal group
When carbonomonoxide concentration is 0) it is low, except for the normal group, without significant difference (p > 0.05) between each processing group.
Embodiment 2
Measurement 44.64mmoL/L carbon monoxide exposes the γ H2AX induced after 15,30,45,60 and 90min respectively.
Experimentation is carried out according to described in embodiment 1, only difference is that, the concentration of contamination of carbon monoxide is fixed on
44.64mmoL/L, contamination time are respectively 0,15,30,45,60 and 90min.
The carbon monoxide that Fig. 4 show 44.64mmoL/L induces A549 thin after 0,15,30,45, the 60 and 90min that contaminates
The when m- effect relation curve for the γ H2AX that born of the same parents generate.As seen from the figure, as contamination time increases, the γ of A549 cell generation
H2AX is compared with normal group and is reduced, and the γ H2AX of exposure induction of each time point is without significant difference (p > 0.05).
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 deforms, and as long as it does not depart from the spirit of the invention, should belong to the model of appended claims of the present invention
It encloses.
Claims (11)
1. a kind of liquid-vapor interface exposure system combines the method that high intension technology quantitative detection carbon monoxide causes DNA Damage,
It the described method comprises the following steps:
1) cell culture: the adherent growth cell in logarithmic growth phase is seeded in bottom using single cell suspension and is filtered as permeability
In the device of film, then described device is carried out to cell culture in cell culture fluid, makes cell single layer on permeable membrane
Growth；
2) cell is contaminated: removing the cell culture fluid in described device, withdrawing device is placed in comprising cell contamination liquid and toxicity
In the contamination storehouse of gas, so that the permeable membrane bottom in device is contacted with cell contamination liquid, while on permeable membrane
Cell surface is exposed to poisonous gas up to 1h, wherein cell contamination liquid is identical as the cell culture fluid in step 1), and
Wherein the poisonous gas is made of air and carbon monoxide and wherein the concentration of carbon monoxide is > 0 and≤44.64mmoL/L,
The flow of the poisonous gas is 20mL/min；
3) immunofluorescence label:
Described device 3-1) is taken out from contamination storehouse, washs, is subsequently placed in pallet, poly first is added in the cell into device
Aldehyde carries out cell and fixes；
Described device and pallet 3-2) are washed, TritonX-100 is then added in the cell into device so that cell-permeant；
Described device and pallet 3-3) are washed, seralbumin closing is then added in the cell into device, one is added later
Anti- i.e. anti-γ H2AX antibody is incubated for；
Described device and pallet 3-4) are washed, the secondary antibody that immunofluorescence label is then added in the cell into device is incubated
It educates；
Described device and pallet 3-5) are washed, DAPI is then added in the cell into device and is dyed；
Described device and pallet 3-6) are washed, is saved；
4) high intension technology quantitative detection: the fluoremetry of the nucleus and γ H2AX of the cell is carried out, respectively to be identified
Effective cell effective cell core in the average fluorescent strength that detects characterize the content of γ H2AX.
2. the method according to claim 1, wherein the cell is connect in the step 1) with single cell suspension
Kind in the device that bottom is permeable membrane, then in cell culture fluid in 37 DEG C, 5%CO2Under the conditions of cultivate for 24 hours.
3. the permeable membrane is dacron membrane the method according to claim 1, wherein in the step 1),
The aperture of aperture is 0.4 μm on film.
4. the method according to claim 1, wherein before cell inoculation, being trained with cell in the step 1)
Described device is balanced 1-2h at 37 DEG C by nutrient solution or PBS.
5. the method according to claim 1, wherein in the step 2), carbon monoxide in the poisonous gas
Concentration be 8.93-44.64mmoL/L.
6. the method according to claim 1, wherein in the step 4), the nucleus and γ of the cell
The fluoremetry of H2AX carries out under two groups of different excitation wavelengths and launch wavelength respectively；
Wherein, the step 3-4) in, the secondary antibody that the secondary antibody of immunofluorescence label marks for Alexa Fluor 488, and institute
It states in step 4), carries out the fluoremetry of nucleus with excitation wavelength 358nm and launch wavelength 461nm in channel one, logical
The fluoremetry of γ H2AX is carried out in road two with excitation wavelength 495nm and launch wavelength 519nm, is identified with to obtain channel one
Thus the average fluorescent strength detected of effective cell core internal channel two of effective cell characterizes the content of γ H2AX.
7. according to the method described in claim 6, it is characterized in that, measurement multiple is 100 times, every hole point in two channels
9 visuals field of analysis and measurement.
8. the method according to claim 1, wherein the method also includes setting does not have in step 3-3)
The blank control group that primary antibody is added, secondary antibody is only added in step 3-4), to obtain being drawn by non-specific adsorption in step 4)
The blank signal risen, thus deducts blank signal from the fluoremetry signal detected.
9. method according to any one of claim 1 to 8, which is characterized in that pass through a variety of packets of setting in step 2)
The poisonous gas of carbon monoxide containing various concentration carries out the method, and the γ H2AX finally obtained according to step 4) is average glimmering
Luminous intensity and the concentration of carbon monoxide draw dose-effect curve.
10. method according to any one of claim 1 to 8, which is characterized in that by contaminating cell in step 2)
The poisonous gas of carbon monoxide of the contact comprising same concentrations reaches different time in malicious storehouse, the method is carried out, finally according to step
The time of the rapid γ H2AX average fluorescent strength 4) obtained and contact toxicity gas m- effect curve when drawing.
11. the method according to claim 1, wherein the poisonous gas passes through 0.2 μm in the step 2)
Enter in the contamination storehouse after membrane filtration.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710156524.3A CN107064481B (en) | 2017-03-16 | 2017-03-16 | A kind of liquid-vapor interface exposure system combines the method for high intension technology quantitative detection carbon monoxide cause DNA Damage |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710156524.3A CN107064481B (en) | 2017-03-16 | 2017-03-16 | A kind of liquid-vapor interface exposure system combines the method for high intension technology quantitative detection carbon monoxide cause DNA Damage |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107064481A CN107064481A (en) | 2017-08-18 |
| CN107064481B true CN107064481B (en) | 2019-07-23 |
Family
ID=59620887
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710156524.3A Active CN107064481B (en) | 2017-03-16 | 2017-03-16 | A kind of liquid-vapor interface exposure system combines the method for high intension technology quantitative detection carbon monoxide cause DNA Damage |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107064481B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140489A (en) * | 2011-01-24 | 2011-08-03 | 中国烟草总公司郑州烟草研究院 | Method for testing cytotoxicity in full smoke contamination of cigarette |
| CN102735804A (en) * | 2012-07-23 | 2012-10-17 | 浙江中烟工业有限责任公司 | Screening method of immunologic function evaluation indexes of anthropopathic nose-only rat |
| CN202671541U (en) * | 2012-05-29 | 2013-01-16 | 云南烟草科学研究院 | Cellular gas and liquid interface contact-type cigarette total-smoke exposure device |
| CN103399159A (en) * | 2013-08-06 | 2013-11-20 | 国家烟草质量监督检验中心 | Quantitative evaluation method for cigarette smoke induced cell DNA damage |
| CN104931473A (en) * | 2015-06-16 | 2015-09-23 | 云南中烟工业有限责任公司 | Evaluation method for measuring cell DNA damage caused by soluble heavy metal |
| CN205139012U (en) * | 2014-05-12 | 2016-04-06 | 赛洛米克斯股份有限公司 | System for carry out automatic high intension cell formation of image |
-
2017
- 2017-03-16 CN CN201710156524.3A patent/CN107064481B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140489A (en) * | 2011-01-24 | 2011-08-03 | 中国烟草总公司郑州烟草研究院 | Method for testing cytotoxicity in full smoke contamination of cigarette |
| CN202671541U (en) * | 2012-05-29 | 2013-01-16 | 云南烟草科学研究院 | Cellular gas and liquid interface contact-type cigarette total-smoke exposure device |
| CN102735804A (en) * | 2012-07-23 | 2012-10-17 | 浙江中烟工业有限责任公司 | Screening method of immunologic function evaluation indexes of anthropopathic nose-only rat |
| CN103399159A (en) * | 2013-08-06 | 2013-11-20 | 国家烟草质量监督检验中心 | Quantitative evaluation method for cigarette smoke induced cell DNA damage |
| CN205139012U (en) * | 2014-05-12 | 2016-04-06 | 赛洛米克斯股份有限公司 | System for carry out automatic high intension cell formation of image |
| CN104931473A (en) * | 2015-06-16 | 2015-09-23 | 云南中烟工业有限责任公司 | Evaluation method for measuring cell DNA damage caused by soluble heavy metal |
Non-Patent Citations (2)
| Title |
|---|
| Detection of the cytotoxicity of water-insoluble fraction of cigarette smoke by direct exposure to cultured cells at an air–liquid interface;Hidenori Nara，et al;《Experimental and Toxicologic Pathology》;20131231;第65卷;摘要，第683页右栏第2段至第685页第2.6节 * |
| 迷迭香酸对CHO细胞DNA损伤的防护作用;夭建华 等;《香料香精化妆品》;20160229(第1期);第1.3.2节至第3节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107064481A (en) | 2017-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Perfetto et al. | Amine‐reactive dyes for dead cell discrimination in fixed samples | |
| CN103399159B (en) | Quantitative evaluation method for cigarette smoke induced cell DNA damage | |
| CN101874206B (en) | Method for end-titre determination and the evaluation thereof by means of an indirect immunofluorescence assay | |
| US20090029378A1 (en) | High sensitivity multiparameter method for rare event analysis in a biological sample | |
| US20090317796A1 (en) | Indirect immunofluorescence assay typing kit for coxsackievirus A group and method for typing coxsackievirus A group | |
| CN102713626A (en) | Immunochemical detection of single target entities | |
| CN105527430A (en) | Kit for directly detecting 71 type enterovirus and A16 type coxsackievirus and application thereof | |
| CN107121553B (en) | A kind of liquid-vapor interface exposure system combines the method for high intension technology quantitative detection 1,3- butadiene cause DNA Damage | |
| Kudłak et al. | Revision of biological methods for determination of EDC presence and their endocrine potential | |
| Dellavance et al. | Detection of autoantibodies by indirect immunofluorescence cytochemistry on HEp-2 cells | |
| CN112924669B (en) | Multicolor flow measurement immunochromatography test strip based on three primary colors of optics and preparation and detection methods thereof | |
| Strovas et al. | Direct measurement of oxygen consumption rates from attached and unattached cells in a reversibly sealed, diffusionally isolated sample chamber | |
| CN102735836A (en) | Visual rapid combined measuring method of plant pathogen and antibody chip | |
| CN107064481B (en) | A kind of liquid-vapor interface exposure system combines the method for high intension technology quantitative detection carbon monoxide cause DNA Damage | |
| CN106970051A (en) | A kind of method that DNA Damage is caused based on high intension technology quantitative analysis tar | |
| CN105606802A (en) | Kit for simultaneously detecting eight pig disease related antibodies and use method of kit | |
| CN106996928A (en) | A kind of magnetic bead chemical luminescence reagent kit for detecting tuberculosis infection and its application | |
| CN107132344A (en) | A kind of method that DNA Damage is caused based on high intension technology quantitative analysis aldehyde material | |
| CN107153054A (en) | A kind of method that DNA Damage is caused based on high intension technology quantitative analysis nicotine | |
| Campbell et al. | Rapid antigen tests | |
| Fedr et al. | Variability of fluorescence intensity distribution measured by flow cytometry is influenced by cell size and cell cycle progression | |
| CN112945919B (en) | Method and system for detecting virus neutralizing antibody and application thereof | |
| CN112858243B (en) | Method for visually monitoring cytochrome c in living cell and evaluating vomitoxin cytotoxicity | |
| Bradwell et al. | Immunofluorescent antinuclear antibody tests | |
| Broccolo et al. | A rapid semi-quantitative test for determination of SARS-CoV-2 antibody levels |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |