CN103834715B - WST-8 (water soluble tetrazolium-8) testing method of in vitro cytotoxicity of tobacco juice of electronic cigarette - Google Patents
WST-8 (water soluble tetrazolium-8) testing method of in vitro cytotoxicity of tobacco juice of electronic cigarette Download PDFInfo
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- CN103834715B CN103834715B CN201410094934.6A CN201410094934A CN103834715B CN 103834715 B CN103834715 B CN 103834715B CN 201410094934 A CN201410094934 A CN 201410094934A CN 103834715 B CN103834715 B CN 103834715B
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Abstract
The invention relates to a WST-8 (water soluble tetrazolium-8) testing method of in vitro cytotoxicity of a tobacco juice of an electronic cigarette. The method comprises the following steps: 1) preparation of an experimental reagent; 2) cell inoculation; 3) contaminating of the tobacco juice of the electronic cigarette; 4) dyeing by WST-8; and 5) result and analysis. Compared with the prior art, the method provided by the invention has the characteristics that the testing step of washing the cells for many times in the whole testing process is not required, so that the operation is simpler and more convenient; the step of changing a culture solution is cancelled during WST-8 dyeing, so that a diluted culture solution can be directly added to react; formazan generated by the WST-8 is water soluble, so that the subsequent dissolving step is canceled, and the light absorption value detection can be realized within 1.5-2 hours after dyeing; the testing period is short and the testing method is convenient and fast. As the WST-8 is non-toxic to the cells, the testing method has the advantages of no damage to the cells, high detection flux, high sensitivity, accurate quantification and the like.
Description
Technical field
The present invention relates to the mensuration of tobacco juice for electronic smoke vitro cytotoxicity, is based on WST-8(water-soluble tetrazolium salts specifically) method for quantitatively determining of cell survival rate after the contamination of Dye Analysis tobacco juice for electronic smoke.
Background technology
Electronic cigarette has another name called electronics Nicotine transfer system.The official definition of the World Health Organization to electronics Nicotine transfer system is: electronics Nicotine transfer system is a kind of consuming product, can transmit Nicotine to lung.Electronic cigarette is generally all made up of three parts: tobacco rod, spraying gun, tobacco juice.In recent years, the novel products such as electronic cigarette are increased rapidly on market, various countries, and the consumer group constantly expands.Some electronic cigarette product also declares that it is less than the hazardness of traditional cigarette.But less to the toxicity assessment research of electronic cigarette at present, correlative study only uses the cytotoxicity of mtt assay to tobacco juice for electronic smoke or smog to evaluate, and result shows that the cytotoxicity of different tobacco juice for electronic smoke differs greatly.But due to MTT through the bluish voilet crystallisate that the enzymatic conversion of viable cell mitochondrial dehydrogenase is produced need add methyl-sulphoxide dissolve after can colorimetric detection, complex operation step, easily impacts experimental result.And WST-8(water-soluble tetrazolium salts) dyestuff is a kind of fast high-sensitive degree detection reagent being applied to cell proliferation and cell survival rate, deposit in case at electron coupling reagent, orange-yellow formazan can be generated by more Intramitochondrial desaturase reduction.Cell proliferation is more, then color is darker; Cell survival rate is lower, then color is more shallow.It is water miscible that WST-8 produces formazan, can save dissolving step follow-up in the methods such as MTT.Compared with the more early stage WST-1 dyestuff that it is of the same type, WST-8 dye stability is stronger, without the need to cryopreservation.Highly sensitive, solvability is stronger.WST-1 dyestuff often needs to add toughener to improve response signal in use, and produces certain toxicity to cell.And use WST-8 dyestuff without the need to adding toughener, and to cell without overt toxicity, make detected result more accurate.In addition, because WST-8 dyestuff is to cytotoxic, cell still can carry out other toxicological experiments such as toluylene red absorption after using WST-8 dyestuff to carry out cytotoxicity detection.And by changing normal cell substratum, realize cultivating the continuation of test cell.
The cytotoxicity of tobacco juice for electronic smoke is lower, when carrying out Cytotoxic evaluation, needs highly sensitive test method; When carrying out the toxotest of a large amount of electronic cigarette sample and multiple Biological indicators, also require that testing method is quick, easy and better compatible.
Summary of the invention
Object of the present invention is intended to overcome prior art defect, and based on the principle that WST-8 dye colour reacts, according to the Cytotoxic feature of tobacco juice for electronic smoke, the measuring method of a kind of tobacco juice for electronic smoke vitro cytotoxicity of foundation.There is the advantages such as harmless to cell, detection flux is high, highly sensitive, quantitatively accurate.
The object of the invention is to be achieved through the following technical solutions:
A kind of tobacco juice for electronic smoke vitro cytotoxicity WST-8 testing method, comprises the following steps:
1) solution preparation:
(1) cell culture medium: solvent is RPMI-1640 substratum, wherein the volume percent of foetal calf serum is 10%, and the concentration of L-glutaminate is 2 mM, and the concentration of penicillin is 100 IU/ml, and the concentration of Streptomycin sulphate is 100 μ g/ml.
(2) tobacco juice for electronic smoke contamination solution:
Because the main moiety of tobacco juice for electronic smoke comprises the humectant such as propylene glycol, glycerol, cause tobacco juice for electronic smoke solution density comparatively large, accurately cannot pipette the preparation that volume carries out contamination solution.Therefore, in the present invention service precision be not less than ten thousand/ analytical balance precise is carried out to tobacco juice for electronic smoke, then use cell culture media solution preparation maximum concentration contamination solution (100mg/ml), all the other contamination solution obtain by using cell culture media solution to carry out dilution to maximum concentration contamination solution.
(3) WST-8 dye liquor: 4 DEG C of preservations.
(4) positive control: sodium lauryl sulphate (SDS) (200 μ g/ml, 100% cell lethality concentration).
2) process of cell sample:
(1) cell inoculation: select attached cell, comprise Chinese hamster ovary cell CHO, human lung cancer cell A549 and people's lung normal epithelium cell BEAS-2B cell.Because the multiplication rate of different clone is different, experimentally cell category used is needed to be optimized cell-seeding-density.Then inoculate the 100ul cell suspension containing best inoculum density number of cells respectively to 96 orifice plates (except most peripheral 36 hole), Tissue Culture Plate is put into cell culture incubator and hatch 24 hours;
(2) tobacco juice for electronic smoke contamination: after cell cultures 24 h, suck nutrient solution, 96 orifice plates (except most peripheral 36 hole) often row 6 hole are one group and are set to blank group, tobacco juice for electronic smoke contamination group and positive controls process respectively.Each group adds 100 μ l cell culture mediums, 100 μ l tobacco juice for electronic smoke contamination solution or positive control solution liquid respectively, and the best concentration of contamination of tobacco juice for electronic smoke is 10ug/ml ~ 100 ug/ml, should arrange and be no less than 7 non-zero concentration of contaminations.Select wherein 6 holes to add cell culture medium as substratum ground control in most peripheral 36 holes of 96 orifice plates, then select 6 holes to add the highest tobacco juice for electronic smoke contamination solution as contamination solution ground control, in 37 DEG C, 5% CO
224 h are cultivated under condition.
(3) WST-8 dyeing: tobacco juice for electronic smoke is contaminated after 24 h, and every hole adds the WST-8 dye liquor of 10 μ l through incubation, is placed in 37 DEG C, 5% CO
22h is hatched under condition.Hatch quick oscillation 1 min under rear room temperature, use microplate reader to detect the light absorption value A of every hole at 450 nm wavelength places
450.
Light absorption value A: A=A450 nm
The absorbance values of A (blank group)=6 parallel holes
The absorbance values of A (substratum background)=6 parallel holes
The absorbance values of A (contamination solution background)=6 parallel holes
The absorbance values of A (positive control)=6 parallel hole
Cell survival rate (%)=(A
contamination group-A
contamination solution background)/(A
blank group-A
substratum background)
Note: SDS positive control used is 100% cell-lethal concentration, therefore A
positive controlshould 0.3 be less than or equal to.
In the present invention, before carrying out Cytotoxic evaluation, need be optimized the cell-seeding-density of contamination cell strain used, in 96 orifice plates, inoculation contains the 100ul cell suspension of different number of cells respectively, Tissue Culture Plate is put into cell culture incubator and hatches 24 hours.Then every hole adds the WST-8 dye liquor of 10 μ l through incubation, as 37 DEG C, 5% CO
2hatch under condition.Hatch quick oscillation 1 min under rear room temperature, use microplate reader to detect the light absorption value A of every hole at 450 nm wavelength places
450.Cell is selected to be in the number of cells of Exponential growth stage as best inoculum density.
Figure 1 shows that Chinese hamster ovary celI, A549 cell, the best inoculum density condition optimizing of BEAS-2B cell.As shown in Figure 1, work as Chinese hamster ovary celI, when the inoculum density of A549 cell is equal to or greater than 20000/hole, Growth of Cells reaches plateau, and this shows that cell-seeding-density is too high, occurs restraining effect, and cell cannot continue propagation and growth.When cell-seeding-density is 10000/hole, cell is in Exponential growth stage and response signal is maximum, and therefore, the best inoculum density of Chinese hamster ovary celI and A549 cell is 10000/hole.For BEAS-2B cell, when inoculum density is equal to or greater than 40000/hole, Growth of Cells reaches plateau, and this shows that cell-seeding-density is too high, occurs restraining effect, and cell cannot continue propagation and growth.When cell-seeding-density is 20000/hole, cell is in Exponential growth stage and response signal is maximum, and therefore, the best inoculum density of BEAS-2B cell is 20000/hole.
In the present invention, the incubation time of WST-8 dye liquor is optimized, after Figure 2 shows that Chinese hamster ovary celI different concns tobacco juice for electronic smoke sample A contamination 24h, every hole adds the WST-8 dye liquor of 10 μ l through incubation, be placed in 37 DEG C, hatch 0.5h respectively, 1 h, 1.5h under 5% CO2 condition, 2 h, after 3h, 4h, microplate reader is used to detect the light absorption value of every hole at 450 nm wavelength places.As shown in Figure 2, when WST-8 dye liquor contamination time is 0.5h and 1 h, the absorbance of detection is low and without obvious dose-effect relationship, when WST-8 dye liquor incubation time is 1.5h-2h, concentration of contamination and detection signal have good dose-effect relationship.And when incubation time is 3h and 4h, increase although detect absorbance, dose-effect relationship is deteriorated, and therefore selects the incubation time of WST-8 to be 1.5h-2h.
For the cytotoxicity of accurate evaluation tobacco juice for electronic smoke, in the present invention, the concentration of contamination of tobacco juice for electronic smoke is optimized, Figure 3 shows that Chinese hamster ovary celI uses 10mg/ml respectively, 20mg/ml, 30mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 80 mg/ml, electronic cigarette sample 1 tobacco juice of 100 mg/ml is contaminated the dose effect curve obtained, as shown in Figure 3, when concentration of contamination is 10mg/ml-100 mg/ml, good dose effect curve can be obtained, 10mg/ml-100 mg/ml scope should be comprised between the chemical contaminated zone therefore investigating tobacco juice for electronic smoke cytotoxic concentration.
Hatch whether have cytotoxicity for investigating WST-8 dye liquor, and with the compatibility of other toxicological experiments such as toluylene red absorption.Carried out two groups of simultaneous tests in the present invention, in battery of tests, use tobacco juice for electronic smoke 1 couple of Chinese hamster ovary celI contamination 24h, every hole adds the WST-8 dye liquor of 10 μ l through incubation, hatches 2h, uses microplate reader to detect the light absorption value of every hole at 450 nm wavelength places simultaneously.Then carry out toluylene red absorption test according to patent [CN 102140489 A] method, use the light absorption value of microplate reader detection at 540 nm wavelength places, calculate relative light absorption value and cell survival rate.In second group of test, use tobacco juice for electronic smoke 1 pair of Chinese hamster ovary celI contamination 26h, then directly carry out toluylene red absorption test according to patent [CN 102140489 A] method, calculate relative light absorption value and cell survival rate.The result of two groups of tests as shown in Figure 4, result shows, the red absorption test result of WST-8 staining procedure centering has no significant effect, prove that WST-8 dye liquor is without obvious cytotoxicity, and carrying out after WST-8 tests the detection that also can carry on toluylene red absorption, test Viola crystallina or other biological index.
Accompanying drawing explanation
Fig. 1. cell-seeding-density optimization
Fig. 2. the Cytotoxic dose-effect relationship curve of tobacco juice for electronic smoke under the different incubation time of WST-8
Fig. 3. the Cytotoxic dose-effect relationship curve of electronic cigarette brand 1 tobacco juice
Fig. 4. the cytotoxicity testing experiment of WST-8 dye liquor
Fig. 5. the Cytotoxic dose-effect relationship curve of electronic cigarette brand 2 tobacco juice.
Embodiment
The present invention is described further below in conjunction with embodiment, but is not restriction the present invention.
For investigating the cytotoxicity of electronic cigarette sample 2 tobacco juice contamination to Chinese hamster ovary celI, after the Chinese hamster ovary celI of amplification cultivation digests, prepare cell suspension (1 × 10
5individual/ml).Every hole (except most peripheral 36 holes) of 96 orifice plates adds 100 μ l cell suspensions, and inoculum density is 10000/hole, in 37 DEG C, 5% CO
224 h are cultivated under condition.In most peripheral 36 holes, select 6 holes to add 100 μ l cell culture mediums and contrast as substratum, 6 holes add the highest contamination solution of 100 μ l as contamination ground control.After cell cultures 24 h, suck nutrient solution, 96 orifice plates (except most peripheral 36 hole) often row 6 hole are one group and are set to blank group, 8 various dose (5,10 respectively, 20,30,40,50,60,80,100mg/ml) tobacco juice for electronic smoke contamination group and positive control treatment group.Often organize and add 100 μ l cell culture mediums, tobacco juice for electronic smoke contamination solution or positive control respectively, in 37 DEG C, 5% CO
224 h are cultivated under condition.Then every hole adds the WST-8 dye liquor of 10 μ l through incubation, and cell is in 37 DEG C, 5% CO
22 h are hatched under condition.Hatch quick oscillation 1 min under rear room temperature, use microplate reader to detect the light absorption value of every hole at 450 nm wavelength places.As shown in Figure 5, tobacco juice for electronic smoke concentration of contamination and WST-8 detect absorbance good dose-effect relationship to acquired results, by formulae discovery IC50 value.
Claims (2)
1. a tobacco juice for electronic smoke vitro cytotoxicity WST-8 testing method, is characterized in that: comprise the following steps:
1) solution preparation:
(1) cell culture medium: solvent is RPMI-1640 substratum, wherein the volume percent of foetal calf serum is 10%, and the concentration of L-glutaminate is 2 mM, and the concentration of penicillin is 100 IU/ml, and the concentration of Streptomycin sulphate is 100 μ g/ml;
(2) tobacco juice for electronic smoke contamination solution:
Carry out precise with analytical balance to tobacco juice for electronic smoke, then use cell culture media solution preparation maximum concentration contamination solution 100mg/ml, all the other contamination solution obtain by using cell culture media solution to carry out dilution to maximum concentration contamination solution;
(3) WST-8 dye liquor: 4 DEG C of preservations;
(4) positive control: sodium lauryl sulphate (SDS), 200 μ g/ml, 100% cell lethality concentration;
2) cell sample process:
(1) cell inoculation: select attached cell, comprise Chinese hamster ovary cell CHO, human lung cancer cell A549 and people's lung normal epithelium cell BEAS-2B cell, because the multiplication rate of different clone is different, experimentally cell category used is needed to be optimized cell-seeding-density, then except most peripheral 36 hole, inoculate the 100ul cell suspension containing best inoculum density number of cells respectively to 96 orifice plates, Tissue Culture Plate is put into cell culture incubator and hatch 24 hours;
(2) tobacco juice for electronic smoke contamination: after cell cultures 24 h, suck nutrient solution, 96 orifice plates except most peripheral 36 hole often row 6 hole be one group and be set to blank group respectively, tobacco juice for electronic smoke contamination group and positive controls process, each group adds hand-hole respectively and adds 100 μ l cell culture mediums, 100 μ l tobacco juice for electronic smoke contamination solution and positive control SDS solution, tobacco juice for electronic smoke should arrange and be no less than 7 non-zero concentration of contaminations, 10mg/ml-100 mg/ml scope should be comprised between the chemical contaminated zone of tobacco juice for electronic smoke cytotoxic concentration, wherein 6 holes are selected to add cell culture medium as substratum ground control in most peripheral 36 holes of 96 orifice plates, selecting 6 holes to add maximum concentration is again that the tobacco juice for electronic smoke contamination solution of 100mg/ml is as contamination solution ground control, in 37 DEG C, 5% CO
224 h are cultivated under condition,
(3) WST-8 dyeing: tobacco juice for electronic smoke is contaminated after 24 h, and every hole adds the WST-8 dye liquor of 10 μ l through incubation, is placed in 37 DEG C, 5% CO
2hatch 1.5-2h under condition, hatched quick oscillation 1 min under rear room temperature, use microplate reader to detect the light absorption value A of every hole at 450 nm wavelength places
450;
(4) results and analysis:
Light absorption value A: A=A
450 nm
The absorbance values of A (blank group)=6 parallel holes
The absorbance values of A (substratum background)=6 parallel holes
The absorbance values of A (contamination solution background)=6 parallel holes
The absorbance values of A (positive control)=6 parallel hole
Cell survival rate (%)=(A
contamination group-A
contamination solution background)/(A
blank group-A
substratum background)
Note: SDS positive control used is 100% cell-lethal concentration, therefore A
positive controlshould 0.3 be less than or equal to.
2. tobacco juice for electronic smoke vitro cytotoxicity WST-8 testing method according to claim 1, it is characterized in that: before carrying out Cytotoxic evaluation, need be optimized the cell-seeding-density of contamination cell strain used, the best inoculum density of Chinese hamster ovary celI and A549 cell is 10000/hole, and the best inoculum density of BEAS-2B cell is 20000/hole.
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| CN101551382A (en) * | 2009-05-12 | 2009-10-07 | 镇江旋光生化有限公司 | Cell viability detection kit and preparation method and application thereof |
| CN102618619B (en) * | 2012-03-30 | 2013-09-18 | 中国烟草总公司郑州烟草研究院 | Method for testing in vitro cytotoxicity of cigarette smoke |
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| CN101551382A (en) * | 2009-05-12 | 2009-10-07 | 镇江旋光生化有限公司 | Cell viability detection kit and preparation method and application thereof |
| CN102618619B (en) * | 2012-03-30 | 2013-09-18 | 中国烟草总公司郑州烟草研究院 | Method for testing in vitro cytotoxicity of cigarette smoke |
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