CN103808678A - Method and kit for measuring cell activity and application of kit - Google Patents
Method and kit for measuring cell activity and application of kit Download PDFInfo
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- CN103808678A CN103808678A CN201410064711.5A CN201410064711A CN103808678A CN 103808678 A CN103808678 A CN 103808678A CN 201410064711 A CN201410064711 A CN 201410064711A CN 103808678 A CN103808678 A CN 103808678A
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Abstract
The invention discloses a method and a kit for measuring the cell activity, and application of the kit. The method comprises the following steps: adding a 2,3,5- triphenyltetrazolium chloride (TTC, with the molecular formula of C19H15C1N4) solution into a cell culture system with cells to be measured, performing co-incubation to obtain red formazan crystal, further dissolving the red formazan crystal into dimethyl sulfoxide (DMSO) to obtain a formazan solution, measuring the absorbancy OD value of the formazan solution by using a microplate reader, and calculating to obtain the cell activity indexes such as relative survival rate, the rate of increase and the relative rate of increase of the cells. According to method, the TTC reagent is not reacted with an antioxidant in the detection system, so that the detection result is more accurate when being compared with that of an MTT (Thiazolyl Blue) method, an XTT (Dimethoxy Pyrazotol Yellow) method and a WSTs method. The method has the advantages of high accuracy, low pollution, rapid detection speed, low instrument and equipment requirement, low cost and the like, and the measurement result is not affected by the antioxidant in the measuring system, so that the method is applicable to population and application.
Description
Technical field
The invention belongs to the assay method of cell viability in biological technical field, particularly relate to one and utilize 2, 3, 5-triphenyl tetrazolium chloride (TTC) is measured the method for cell viability, the method can be used for measuring the vigor of adherent and suspended culture cell, than existing based on 3-(4, 5-dimethylthiazole-2)-2, 5-diphenyl tetrazole bromine salt (MTT), 3, 3'-[1-(phenylamino acyl group)-3, 4-tetrazole] result that obtains of the method for the mensuration cell viability such as-bis-(4-methoxyl-6-nitro) benzene sulfonic acid sodium salts (XTT) and water-soluble tetrazole (WSTs) is more reliable, be not easy to be subject to the interference of antioxidant in detection system, thereby can obtain science more, objective, the result of cell viability reliably.
Background technology
In Bioexperiment, in order to measure the impact on indexs such as cell survival, propagation, growth inhibition or toxicity of certain stimulation or medicine, often need to measure the vigor of cell.The method of existing mensuration cell viability comprises mtt assay (Mosmann T.Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays.J Immunol Methods, 1983, 65 (1-2): 55-63.), XTT method (Scudiero DA, Shoemaker RH, Paull KD, et al.Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines.Cancer Res.1988.48 (17): 4827-33.) and WSTs method (Ishiyama M, Tominaga H, Shiga M, Sasamoto K, Ohkura Y, Ueno K.A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet.Biol Pharm Bull.1996.19 (11): 1518-20.) etc., all belong to tetrazolium salts reducing process, principle be can free permeate through cell membranes water-soluble tetrazolium salts by the dehydrogenase type Hai Yuan Cheng Za class material in living cells, dead cell can not reduce tetrazolium salts, the growing amount reflection cell viability of Za class material, and be directly proportional to cell quantity, by calculating the relative survival rate that can draw cell, the rate of increase, the indexs such as relative appreciation rate.Wherein, mtt assay is easy and simple to handle, expense is cheap, comparatively conventional in Cell Biology Experiment, and XTT method and WSTs method generation formazan are soluble, it is more convenient to operate, but because the reagent price using in these detection methods is more expensive, degree of accuracy to experiment does not also promote, thereby popularity rate is not very high in Bioexperiment, and the defect of these detection method maximums is that tetrazolium salts used can be detected multiple reductive agent in system (as L-AA, flavone compound, containing the antioxidant of sulfydryl, seralbumin, glutathione S-transferase, nano particle, polyphenol compound) direct-reduction and disturbed, thereby affect the accuracy of data.Therefore, find a kind of method that can compare Accurate Determining cell viability, for judging that exactly cell viability is significant and being worth, avoid the existence due to multiple antioxidant in detection system to cause objectively estimating the vigor of cell.TTC detection method proposed by the invention can overcome this point well.
(2,3,5-triphenyl-2H-tetrazolium chloride is called for short TTC, molecular formula C to 2,3, 5-Triphenyltertrazoliumchloride
19h
15clN
4) be tetrazolium salts conventional in a kind of biological experiment, its mechanism for existing biological experiment is: after TTC and living tissue and cell are hatched, can be generated red formazan (1 by intracellular dehydrogenase type catalytic reduction, 3,5-triphenyl formazan, is called for short TPF, molecular formula C
19h
16n
4) and be deposited in cell and tissue, and it is red that cell and tissue are shown, and dead cell and tissue can not make its reduction, keep original transparent or canescence, its reaction equation is as follows:
In the past in research, TTC does not have the total vitality of subject for detection of zooblast, and be once used to detect vigor (the Lillie RD of vegetable seeds, Conn HJ.HJ Conn's biological stains.1969, 154-162.), it is mainly the ability whether for measuring plant embryo in vegetable seeds with germination, although be also measure living cells in embryo number, but and be different between the cell viability of the detection animal cultured in vitro of paying close attention in the present invention, because this piece be published in 1969 document detect be the germinating capacity of vegetable cell, and be the total vitality of subject that detects cell in the present invention, especially for zooblast, cannot use ' germination ' ability to carry out similar explanation.Meanwhile, in previous literature, whether can not avoid the interference of the reductive agent in detection system to carry out any analysisanddiscusion for TTC detection method.
Summary of the invention
There is the problem of cross reaction in order to overcome the middle reductive agent detecting in reagent and detection system of existing cell viability assay method (as mtt assay, XTT method, WSTs method etc.), the invention provides one and utilize 2,3,5-triphenyl tetrazolium chloride (TTC, molecular formula C
19h
15clN
4) measure the method for cell viability.
The method of mensuration cell viability provided by the invention is by 2,3, 5-Triphenyltertrazoliumchloride (TTC, molecular formula C
19h
15clN
4) solution joins in the cell culture system that contains cell to be measured, obtain red formazan crystal by hatching altogether, again Gai formazan crystal is dissolved in to get formazan solution in dimethyl sulfoxide (DMSO) (DMSO), measure the absorbance OD value of formazan solution (detecting sample) by microplate reader, calculate the cell viability that detects cultured cell in sample according to absorbance OD value.
Wherein, TTC solution is that mass percentage concentration is the PBS solution of 0.5-1.0%.
The absorbance OD value that detects sample is to measure and obtain under wavelength X=485 ± 5nm by microplate reader.
Described cell viability index comprises one or several in relative survival rate, the rate of increase of cell and the relative rate of increase of cell of viable count, cell.
Concrete, TTC solution is added respectively in one group of institute's cultured cell of accurate counting and hatch altogether, measure this group cell formazan solution to be measured absorbance OD value separately, take cell number gradient as X-axis, absorbance OD value is done absorbance OD value-cell number gradient typical curve for Y-axis; From this typical curve, obtain detecting cell count corresponding to the absorbance OD value of sample as viable count.
The relative survival rate of described cell calculates by following formula:
Wherein: drug treating group OD value obtains the absorbance OD value that formazan solution microplate reader is measured under wavelength X for the cell to be measured after drug treating; The absorbance OD value that control group OD value is measured under wavelength X for the formazan solution microplate reader obtaining through PBS cell to be measured after treatment;
Described cell proliferation rate calculates by following formula:
Wherein: the absorbance OD value that the formazan solution microplate reader that after drug treating, after X hour, OD value obtains for cell to be measured after drug treating X hour is measured under wavelength X; The absorbance OD value that the formazan solution microplate reader that before drug treating, OD value obtains for cell to be measured before drug treating is measured under wavelength X;
The relative rate of increase of described cell calculates by following formula:
Wherein: the absorbance OD value that the formazan solution microplate reader that after drug treating, after X hour, OD value obtains for cell to be measured after drug treating X hour is measured under wavelength X; The absorbance OD value that the formazan solution microplate reader that before drug treating, OD value obtains for cell to be measured before drug treating is measured under wavelength X; The blank group absorbance OD value that after X hour, OD value is measured under wavelength X for the formazan solution microplate reader that cell to be measured obtains after PBS processes X hour.
Cell viability detection method of the present invention, specifically comprises the following steps:
1) preparation TTC detects solution: the TTC detection solution that is 0.5-1.0% by 0.01M phosphate buffer preparation mass percentage concentration;
2) detect solution to the TTC that adds 1/10 volume in cell culture system to be measured, at 37 ℃, hatch altogether 4-12 hour, TTC by cell reduction after Sheng Cheng formazan crystal;
3) leave and take formazan crystal, with dimethyl sulfoxide (DMSO) (DMSO) the Rong Xie formazan crystal of archaeocyte nutrient solution volume 1/5-2/5, obtain formazan solution and detect sample solution;
Formazan solution is transferred to ELISA Plate by 4), measures the absorbance OD value that detects sample solution by microplate reader under 485 ± 5nm wavelength;
5) cell count that obtains detecting cell sample from absorbance OD value-cell number gradient typical curve described in claim 5 is as viable count, or calculates the relative survival rate, the rate of increase of cell or the index such as the rate of increase relatively by formula described in claim 6.
In cell viability detection method of the present invention, described cell is adherent or suspension cultured cells; If suspension cell adds TTC detect solution and hatch at 37 ℃ 4 hours.
Another object of the present invention is to provide a kind of kit for detection of cell viability.
Kit for detection of cell viability provided by the present invention, comprises that mass percentage concentration is the TTC detection solution (with the preparation of 0.01M phosphate buffer) of 0.5-1.0%.
Detect for convenience, in described kit, also comprise dimethyl sulfoxide (DMSO) (DMSO).
Adopt above scheme, the invention provides one and utilize 2,3, 5-Triphenyltertrazoliumchloride (TTC, molecular formula C
19h
15clN
4) measure method adherent and suspension cell vigor.The method is to utilize intracellular dehydrogenase type reduction TTC to generate red formazan, and the growing amount of formazan and living cells quantity are linear within the specific limits, can obtain the index such as relative survival rate, the rate of increase and the relative rate of increase of cell by calculating.The TTC reagent using due to the method does not react with antioxidants such as L-AA from detection system, glutathione (GSH), N-acetylcysteins (NAC), therefore the testing result obtaining is more accurate than mtt assay, XTT method and WSTs method etc., can reflect better cell viability, there is important value for researchs such as cell proliferation and cell viability analyses.Cell viability assay method of the present invention is obviously better than prior art, have the high and low pollution of accuracy, detection speed fast, to advantages such as instrument and equipment requirement are low and with low cost, and measurement result is not subject to the interference of antioxidant in mensuration system, be applicable to promotion and application.
Below in conjunction with specific embodiment, the present invention is described in further details.
Accompanying drawing explanation
Fig. 1 is the absorption spectrum of TTC formazan solution between 190-920nm of measuring with ultraviolet-visible spectrophotometer
Fig. 2 is the absorbance of measuring each concentration TTC detection solution group by microplate reader under 490nm wavelength, detects the impact of solution on measurement result to detect the TTC of variable concentrations
Fig. 3 is making scatter diagram matched curve, the funtcional relationship between analysis of cells quantity and absorbance, and analysis result shows that the absorbance of cell quantity and mensuration is linear within the specific limits
Fig. 4 is TTC and MTT and the response situation of different antioxidants
Embodiment
In following embodiment, method therefor is conventional method if no special instructions.
Described percent concentration is mass percent concentration if no special instructions, mass/volume (W/V) percent concentration (g/100mL of unit), or concentration of volume percent.
In the listed each embodiment of the present invention, same material and reagent are general if no special instructions.The approach that obtains of the various biomaterials that are described in embodiment is only to provide approach that a kind of experiment obtains to reach concrete disclosed object, should not become the restriction to biological material source of the present invention.In fact, the source of biomaterial used is widely, any keep on the right side of the law and the moral ethics biomaterial that can obtain can be replaced and use according to the prompting in embodiment.
Embodiment implements under take technical solution of the present invention as prerequisite, has provided detailed embodiment and concrete operating process, and embodiment will contribute to understand the present invention, but protection scope of the present invention is not limited to following embodiment.
The technical essential of the present embodiment:
A) TTC detects preparation and the using method of solution;
B) dimethyl sulfoxide (DMSO) (DMSO) dissolves the absorption spectrum that generates formazan and solution thereof;
C) TTC measures the best working concentration of cell viability.
The present embodiment is determined 2,3, 5-Triphenyltertrazoliumchloride (TTC, molecular formula C
19h
15clN
4) whether can be used for measuring cell viability and working concentration, carry out following operation:
1) preparation TTC detects solution: with 0.01M phosphate buffer (PBS, formula: 137mM NaCl, 2.7mM KCl, 10mM Na
2hPO
4, 2mM KH
2pO
4, pH7.2-7.4) and dissolve TTC medicine, be mixed with mass percentage concentration and be respectively 0.1%, 0.2%, 0.5%, 1.0%, 1.5% and 2.0% TTC and detect solution.Be the filtrator filtration sterilization of 0.22 μ m with aperture, packing ,-20 ℃ of preservations.
2) (male Rattus norvegicus Adrenal Pheochromocytoma, derives from ATCC, article No.: ATCC to PC12 cell
cRL-1721
tM) be inoculated in 24 orifice plates after digestion, with the DMEM in high glucose nutrient culture media containing 5% hyclone, 5% horse serum, 100U/mL penicillin, 100U/mL streptomysin, (formula is referring to the GIBCO of Life Technologies company
12800-017), in 37 ℃, 5% CO2gas incubator, cultivate.In the time that cell density reaches approximately 80%, by the volume of cell culture fluid 1/10, (50 μ l) add the TTC of variable concentrations to detect solution, continue to cultivate 4 hours (4-12 hour all can) in incubator, TTC by cell reduction after Sheng Cheng formazan crystal.
3) outwell nutrient solution, add the dimethyl sulfoxide (DMSO) (DMSO) of original fluid volume 2/5 (200 μ l, 1/5-2/5 volume all can) to dissolve and generate formazan crystal.
4) get 100 μ l formazan solution to ultraviolet-visible spectrophotometer (Varian Cary50) and scan the absorption spectrum in 190-920nm wavelength coverage.Result, shown in Fig. 1 (horizontal ordinate represents wavelength, and ordinate represents absorbance), obtains the maximum absorption band of TTC formazan solution at 485nm place, can use this wavelength or near wavelength to measure while showing to measure cell viability.
5) transfer to ELISA Plate obtaining formazan solution through variable concentrations TTC detection solution-treated PC12 cell, absorbance by microplate reader (BioRad680) in 490nm wavelength (the detection wavelength that approaches 485nm all can use, and the present embodiment is selected 490nm) the each concentration group of lower mensuration.
Result as shown in Figure 2, can be found out, generates formazan content the highest by mass percentage concentration when the TTC that is 0.5-1.0% detection measured in solution cell viability, is suitable for measuring cell viability.
The technical essential of the present embodiment:
A) the suitable cell quantity of measuring cell viability with TTC is 4 × 10
4-2 × 10
5in scope;
B) make scatter diagram matched curve, the funtcional relationship between absorbance and cell quantity that analysis TTC method obtains.
The present embodiment is determined 2,3, 5-Triphenyltertrazoliumchloride (TTC, molecular formula C
19h
15clN
4) measure the absorbance of cell viability gained and the relation of cell quantity, carry out following operation:
1) preparation TTC detects solution: with 0.01M phosphate buffer (PBS, formula: 137mM NaCl, 2.7mM KCl, 10mM Na
2hPO
4, 2mM KH
2pO
4, pH7.2-7.4) and dissolve TTC medicine, be mixed with mass percentage concentration and be 1.0% TTC and detect solution.Be the filtrator filtration sterilization of 0.22 μ m with aperture, packing ,-20 ℃ of preservations.
2) the DMEM in high glucose nutrient culture media containing 5% hyclone, 5% horse serum, 100U/mL penicillin, 100U/mL streptomysin (formula is referring to the GIBCO 12800-017 of Life Technologies company) for PC12 cell is cultivated in 37 ℃, 5% CO2gas incubator.In the time that cell density reaches 80%-90%, digest and use blood counting chamber counting cells, respectively by 4 × 10
4, 8 × 10
4, 1.2 × 10
5, 1.6 × 10
5, 2 × 10
5cell number inoculating cell to uncovered centrifuge tube, by cell culture fluid 1/10 volume, (10 μ l) add 1.0% TTC to detect solution in PC12 cell, continue to cultivate 4 hours (4-12 hour all can) in incubator, TTC by cell reduction after Sheng Cheng formazan crystal.
3) centrifugal collecting cell is with formazan crystal is outwelled supernatant nutrient solution, add 100 μ l(1/5-2/5 volumes all can) dimethyl sulfoxide (DMSO) (DMSO) dissolve and generate formazan.
4) obtain formazan solution and transfer to ELISA Plate detect solution-treated PC12 cell through 1.0%TTC, with microplate reader (BioRad680) mensuration absorbance under 490nm wavelength (the detection wavelength that approaches 485nm all can use, and the present embodiment is selected 490nm).Make scatter diagram matched curve, the funtcional relationship between analysis of cells quantity and absorbance.
Result as shown in Figure 3, can be found out, when cell quantity is 4 × 10
4-2 × 10
5scope in time, with TTC measure cell viability data (formazan solution absorbance value) and cell quantity between linear, using this curve as TTC for PC12 cytoactive detect absorbance OD value-cell number gradient typical curve.
Embodiment 3, utilize TTC measure cell viability
One, testing sample 1: medicine superoxol is processed PC12 cell
The present embodiment utilizes 2,3, 5-Triphenyltertrazoliumchloride (TTC, molecular formula C
19h
15clN
4) PC12 cell viability in working sample.Operating process is as follows:
1) preparation TTC detects solution: with 0.01M phosphate buffer (PBS, formula: 137mM NaCl, 2.7mM KCl, 10mM Na
2hPO
4, 2mM KH
2pO
4, pH7.2-7.4) and dissolve TTC medicine, the TTC of (0.5-1.0% all can) detects solution to be mixed with mass percentage concentration and to be 1.0%.Be the filtrator filtration sterilization of 0.22 μ m with aperture, packing ,-20 ℃ of preservations.
2) after PC12 cell dissociation, be inoculated in 24 orifice plates, with the DMEM in high glucose nutrient culture media containing 5% hyclone, 5% horse serum, 100U/mL penicillin, 100U/mL streptomysin, (formula is referring to the GIBCO of Life Technologies company
12800-017), in 37 ℃, 5% CO2gas incubator, cultivate, in the time that cell density reaches approximately 80%, change fresh DMEM in high glucose nutrient culture media and add 1mM hydrogen peroxide (medicine) to process 2 hours (blank group adds the PBS of same volume) of PC12 cell, rear recovery DMEM in high glucose is cultivated, and cultured cells is divided into groups.
Experimental group (drug treating group-experimental group): by the volume of cell culture fluid 1/10 (50 μ l) add 1.0% TTC to detect solution in PC12 cell, TTC by cell reduction after Sheng Cheng formazan crystal.
Comparative group (drug treating group-comparative group): measure cell viability as comparing with reference to method with traditional mtt assay, (50 μ are 0.5% MTT solution (0.5g MTT prepares in being dissolved in PBS) l) in identical grouping, to add 1/10 volume, continue to cultivate 4 hours (4-12 hour all can) in incubator, MTT is by the rear corresponding formazan crystal (MTT and TTC are incomplete same by the rear of generation of cell reduction Za class, but are all called formazan) that generates of cell reduction.
3) outwell nutrient solution, (the 200 μ l) dimethyl sulfoxide (DMSO) (DMSO) of (1/5-2/5,100 μ l-200 μ l all can) dissolve and generate formazan crystal, obtain formazan solution separately to add original fluid volume 2/5.
4) Ge formazan solution is transferred to ELISA Plate, by microplate reader (BioRad680) mensuration absorbance OD value under 490nm wavelength (wavelength that TTC formazan is used), reference method (mtt assay) comparative sample is in the lower absorbance OD value of measuring of 570nm wavelength (near the wavelength that MTT formazan is used, MTT formazan has maximum absorption band this wavelength).Data are recorded in table 1.
According to measured OD value, obtain viable count from absorbance OD value-cell number gradient typical curve (referring to Fig. 3 embodiment 2).
As blank (control group), calculate the relative survival rate of cell with PBS according to surveyed OD value, computing formula is as follows:
Wherein: medicine (hydrogen peroxide) processed group OD value is cell absorbance OD value that formazan solution microplate reader is measured under 490nm wavelength that obtains after medicine hydrogen peroxide treatment; The absorbance OD value that the formazan solution microplate reader that control group OD value obtains after PBS processes for cell is measured under 490nm wavelength.
Measurement result: referring to table 1.
Table 1: medicine superoxol is processed PC12 cell detection results
| ? | Viable count (105) | Relative survival rate | OD value |
| Blank group (MTT) | 10.184 | - | 0.6952±0.0086 |
| Hydrogen peroxide group (MTT) | 5.818 | 37.18% | 0.2586±0.0057 |
| Blank group (TTC) | 10.212 | - | 0.7032±0.0078 |
| Hydrogen peroxide group (TTC) | 5.884 | 37.71% | 0.2652±0.0051 |
Analyze knownly, mtt assay and TTC method all can be carried out cell viability mensuration to the PC12 cell of hydrogen peroxide treatment, and the relative survival rate of the two and OD are worth measurement result close.Conclusion: adding medicine in testing sample 1 is hydrogen peroxide, and cell is had damage.In addition, TCC measurement result and MTT measurement result are coincide (in this experimental cell system, do not have the interference of antioxidant, MTT measurement result can be regarded as accurate result), show that accuracy of the present invention is better.
Two, testing sample 2: medicine epidermal growth factor is processed PC12 cell
Operating process and aforementioned 1)-4) identical, wherein step 2) use epidermal growth factor purchased from Sigma company (U.S.), working concentration is 20nM.Cultured cells is carried out following grouping:
Group before drug treating: when cell density reaches approximately 80%, change fresh DMEM in high glucose nutrient culture media, synchronize and cultivate with drug treating group.
Drug treating group: in the time that cell density reaches approximately 80%, change fresh DMEM in high glucose nutrient culture media, and adding the epidermal growth factor drug treating PC12 cell 2 hours of 20nM, rear recovery DMEM in high glucose is cultivated, and cultured cells is divided into TTC drug treating group and MTT drug treating group.
Blank group: in the time that cell density reaches approximately 80%, change fresh DMEM in high glucose nutrient culture media, and add PBS to process PC12 cell 2 hours, rear recovery DMEM in high glucose is cultivated, as the blank of drug treating group.
Measure each group of absorbance OD value.Measurement result is recorded in table 2.
By calculating below the rate of increase of cell:
Wherein: the absorbance OD value that the formazan solution microplate reader that after drug treating, after X hour, OD value obtains after drug treating X hour for cell is measured under 490nm wavelength (mtt assay is at 570nm); The absorbance OD value that the formazan solution microplate reader that before drug treating, OD value obtains before drug treating for cell is measured under 490nm wavelength.
The relative rate of increase of cell calculates by following formula:
Wherein: the absorbance OD value that the formazan solution microplate reader that after drug treating, after X hour, OD value obtains after drug treating X hour for cell is measured under 490nm wavelength (mtt assay is at 570nm); The absorbance OD value that the formazan solution microplate reader that before drug treating, OD value obtains before drug treating for cell is measured under 490nm wavelength; The absorbance OD value that the blank group formazan solution microplate reader that after X hour, OD value obtains after PBS processes X hour for cell is measured under 490nm wavelength.
For testing sample 2 measurement results in table 2.
Table 2 medicine epidermal growth factor is processed PC12 cell detection results
| ? | Cell proliferation rate | The rate of increase relatively | OD value |
| Blank group (mtt assay) | - | - | 0.6006±0.0087 |
| Group (mtt assay) before drug treating | 13.06% | 131.92% | 0.5392±0.0364 |
| Epidermal growth factor subgroup (mtt assay) | - | - | 0.6202±0.0271 |
| Blank group (TTC method) | - | - | 0.6080±0.0114 |
| Group (TTC method) before drug treating | 14.38% | 150.00% | 0.5468±0.0363 |
| Epidermal growth factor subgroup (TTC method) | - | - | 0.6386±0.0272 |
Analyze knownly, use the amount of survival of the characterize cells that mtt assay and TTC method all can be correct after medicine.Because medicine (epidermal growth factor) has proliferation function to cell, can increase Growth of Cells, after drug treating, cell number is higher than blank group, and the relative rate of increase therefore calculating according to formula is all greater than 100%.Show on the other hand, the cell proliferation rate that in this experiment, TTC method calculates with mtt assay and the relative rate of increase are suitable.
The response situation of embodiment 4, TTC and MTT and different antioxidants
The technical essential of the present embodiment:
A) preparation of antioxidant solution;
B) reaction system of TTC and MTT and antioxidant.
In Bioexperiment, cell is subject to after exogenous stimulation or drug effect, conventionally its vigor declines or is dead, often add antioxidant intervention, and a lot of medicines itself are exactly antioxidant or have antioxidation activity, therefore the present embodiment has been measured the situation that MTT and TTC react with Partial Antioxidation agent respectively.
Detect the response situation of TTC and MTT and different antioxidants, carry out following operation:
1) phosphate buffer of use 0.01M (PBS, formula: 137mM NaCl, 2.7mM KCl, 10mM Na
2hPO
4, 2mM KH
2pO
4, pH7.2-7.4) and dissolve respectively glutathione (GSH), N-acetylcystein (NAC) and L-AA, be mixed with the solution that concentration is 100mM.Be the filtrator filtration sterilization of 0.22 μ m with aperture, packing ,-20 ℃ of preservations.
2) in ELISA Plate, dilute respectively GSH, NAC and the L-AA solution of 100mM with DMSO, respectively be mixed with 0.5mM (100 μ solution l), (10 μ are 1.0% TTC solution or 0.5% MTT solution (formula: 0.5g MTT is dissolved in 100mL PBS) l) to add respectively 1/10 volume, at 37 ℃, react 4h, PBS is as the negative control of antioxidant group.
3) by microplate reader (BioRad680), at 490nm, (the detection wavelength that approaches 485nm all can use, the present embodiment is selected 490nm) or 570nm (maximum absorption band of MTT formazan solution is at 554nm, and the present embodiment uses near the wavelength 570nm it to measure) wavelength under measure each group of absorbance.
Result as shown in Figure 4, show that TTC does not react with GSH, NAC and L-AA substantially, and MTT and this three kinds of antioxidants has kickback.Therefore, TTC of the present invention detects the interference that can effectively avoid multiple antioxidant in detection system, can compare the cell viability in Accurate Determining system.
Embodiment 5, for detection of kit and the application thereof of cell viability
According to above embodiment, the present invention can form a kind of kit for detection of cell viability.This kit comprises that mass percentage concentration is TTC detection solution (with the preparation of 0.01M phosphate buffer) 2mL and dimethyl sulfoxide (DMSO) (DMSO) 10mL of 0.5-1.0%, and comprises a operational manual that detects.
Utilize reagent in this kit to carry out the process of operation of cell viability detection as follows:
1) in 50 μ l cell culture fluid to be measured, add TTC to detect solution 50 μ l, after formazan crystal to be generated, outwell nutrient solution, then add 100 μ l-200 μ l dimethyl sulfoxide (DMSO) (DMSO) Rong Xie formazan crystal, obtain formazan solution.
Formazan solution is transferred to ELISA Plate by 2), measures absorbance OD value by microplate reader (BioRad680) under 490nm wavelength.
Afterwards, then according to designed experiment, utilize formula (referring to embodiment 3) to calculate cell viability index, comparative survival rate of cells or the rate of increase and/or the relatively rate of increase.
Claims (10)
1. measuring a method for cell viability, is by 2,3, 5-Triphenyltertrazoliumchloride (TTC, molecular formula C
19h
15clN
4) solution joins in the cell culture system that contains cell to be measured, obtain red formazan crystal by hatching altogether, again Gai formazan crystal is dissolved in to get formazan solution in dimethyl sulfoxide (DMSO) (DMSO), measure the absorbance OD value of formazan solution (detecting sample) by microplate reader, calculate the cell viability that detects cultured cell in sample according to absorbance OD value.
2. cell viability detection method according to claim 1, is characterized in that: TTC solution is that mass percentage concentration is the PBS solution of 0.5-1.0%.
3. cell viability detection method according to claim 1 and 2, is characterized in that: under wavelength X=485 ± 5nm, measure the absorbance OD value that detects sample by microplate reader.
4. according to the cell viability detection method described in claim 1 or 2 or 3, it is characterized in that: described cell viability index comprises one or several in relative survival rate, the rate of increase of cell and the relative rate of increase of cell of viable count, cell.
5. cell viability detection method according to claim 4, it is characterized in that: TTC solution is added respectively in one group of institute's cultured cell of accurate counting and hatch altogether, measure this group cell formazan solution to be measured absorbance OD value separately, take cell number gradient as X-axis, absorbance OD value is done absorbance OD value-cell number gradient typical curve for Y-axis; From this typical curve, obtain detecting cell count corresponding to the absorbance OD value of sample as viable count.
6. cell viability detection method according to claim 4, is characterized in that:
The relative survival rate of described cell calculates by following formula:
Wherein: drug treating group OD value obtains the absorbance OD value that formazan solution microplate reader is measured under wavelength X for the cell to be measured after drug treating; The absorbance OD value that control group OD value is measured under wavelength X for the formazan solution microplate reader obtaining through PBS cell to be measured after treatment;
Described cell proliferation rate calculates by following formula:
Wherein: the absorbance OD value that the formazan solution microplate reader that after drug treating, after X hour, OD value obtains for cell to be measured after drug treating X hour is measured under wavelength X; The absorbance OD value that the formazan solution microplate reader that before drug treating, OD value obtains for cell to be measured before drug treating is measured under wavelength X;
The relative rate of increase of cell calculates by following formula:
Wherein: the absorbance OD value that the formazan solution microplate reader that after drug treating, after X hour, OD value obtains for cell to be measured after drug treating X hour is measured under wavelength X; The absorbance OD value that the formazan solution microplate reader that before drug treating, OD value obtains for cell to be measured before drug treating is measured under wavelength X; The blank group absorbance OD value that after X hour, OD value is measured under wavelength X for the formazan solution microplate reader that cell to be measured obtains after PBS processes X hour.
7. according to the cell viability detection method described in claim 5 or 6, it is characterized in that: comprise the following steps:
1) preparation TTC detects solution: the TTC detection solution that is 0.5-1.0% by 0.01M phosphate buffer preparation mass percentage concentration;
2) detect solution to the TTC that adds 1/10 volume in cell culture system to be measured, at 37 ℃, hatch altogether 4-12 hour, TTC by cell reduction after Sheng Cheng formazan crystal;
3) leave and take formazan crystal, with dimethyl sulfoxide (DMSO) (DMSO) the Rong Xie formazan crystal of archaeocyte nutrient solution volume 1/5-2/5, obtain formazan solution and detect sample solution;
Formazan solution is transferred to ELISA Plate by 4), measures the absorbance OD value that detects sample solution by microplate reader under 485 ± 5nm wavelength;
5) cell count that obtains detecting cell sample from absorbance OD value-cell number gradient typical curve described in claim 5 is as viable count, or calculates the relative survival rate, the rate of increase of cell or the index such as the rate of increase relatively by formula described in claim 6.
8. according to the arbitrary described cell viability detection method of claim 1 to 7, it is characterized in that: described cell is adherent or suspension cultured cells; If suspension cell adds TTC detect solution and hatch at 37 ℃ 4 hours.
9. for detection of a kit for cell viability, comprise that mass percentage concentration is the TTC detection solution (with the preparation of 0.01M phosphate buffer) of 0.5-1.0%.
10. kit according to claim 4, is characterized in that: described kit also comprises dimethyl sulfoxide (DMSO) (DMSO).
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| CN109283149A (en) * | 2018-11-02 | 2019-01-29 | 浙江工业大学 | A method of detecting Gibberella fujikuroi activity biomass in gibberellin fermented liquid |
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