CN109406765B - Cell proliferation and toxicity detection kit - Google Patents
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- CN109406765B CN109406765B CN201811234278.XA CN201811234278A CN109406765B CN 109406765 B CN109406765 B CN 109406765B CN 201811234278 A CN201811234278 A CN 201811234278A CN 109406765 B CN109406765 B CN 109406765B
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- 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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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Abstract
The invention discloses a cell proliferation and toxicity detection kit capable of improving detection sensitivity and stability, shortening reaction time and avoiding detection liquid from being leaked, which comprises detection liquid, wherein the detection liquid consists of WST-8, 2-methyl-1, 4-naphthoquinone, a sodium chloride aqueous solution, a potassium chloride aqueous solution and a calcium chloride aqueous solution, and the molar ratio of the WST-8 to the 2-methyl-1, 4-naphthoquinone is 12-18: 1, the concentrations of the sodium chloride aqueous solution, the potassium chloride aqueous solution and the calcium chloride aqueous solution are respectively 8.6g/L, 0.3 g/L and 0.28 g/L; and the reaction termination solution is 0.05-0.15 mol/L HCl.
Description
Technical Field
The invention relates to the technical field of cell biology, in particular to a cell proliferation and toxicity detection kit which can improve detection sensitivity and stability, shorten reaction time and avoid the addition of detection liquid.
Background
Cell proliferation and toxicity detection are basic methods for determining substance toxicity and evaluating drug safety and cell health, and can indicate the proliferation activity of tumor cells, the cell proliferation activity of target gene transient/stable cell lines, and the gene function research of target gene overexpression or RNAi interference, and play an important role in drug screening and functional safety research. Therefore, cell proliferation and toxicity detection techniques have been widely used in various research fields such as molecular biology, tumor biology, immunology, genetics, pharmacology, and pharmacokinetics.
At present, cell proliferation detection methods mainly comprise three categories, namely DNA synthesis detection, cell proliferation related antigen detection and metabolic activity detection, and the metabolic activity detection method is the most common method. The early detection method of metabolic activity comprises an MTT method, an XTT method, an MTS method, a WST-1 method and the like, namely, the detection kit respectively contains MTT, XTT, MTS and WST-1 detection liquid. The principle of the MTT (tetramethylazozolium) method is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-violet crystal formazan and deposit the formazan in the cells (dead cells do not have the function), after the crystals are dissolved, the 570nm wavelength absorption value is measured, the MTT crystal formation amount is in direct proportion to the number of cells in a certain cell number range, and the higher the absorbance, the stronger the cell activity and the lower the drug toxicity. Although the MTT method has the advantages of convenient and quick operation, sensitivity, no radioactivity and the like, because formazan products need to be dissolved, result deviation can be caused sometimes because formazan is not completely dissolved or is brought out, so that the repeatability is poor, and the MTT method has cytotoxicity.
The XTT (tetrazole derivative) method is similar to the MTT principle, the XTT is reduced into water-soluble brown yellow formazan under the action of living cell mitochondrial dehydrogenase, and the generation amount of the brown yellow formazan is positively correlated with the number of living cells in the presence of an electronic coupling agent, namely Phenazine Methosulfate (PMS). But XTT itself is not readily soluble and the solution is not stable, thus limiting its use.
The MTS method and the WST-1 method are similar to the XTT principle, require the existence of PMS, but overcome the defects of MTT and XTT, have higher sensitivity and specificity, but MTS or WST-1 needs to be prepared separately from PMS and then proportionally mixed when in use, and increase the complexity of operation.
WST-8[2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulphophenyl) -2H-tetrazolium ] is a water-soluble tetrazolium salt which is developed in recent years and is more recent than WST-1, the using principle of the water-soluble tetrazolium salt is similar to that of the method, the water-soluble tetrazolium salt can be reduced into a highly water-soluble orange-yellow formazan product by dehydrogenase in mitochondria in the presence of an electron coupling agent (such as PMS), the amount of the generated formazan is proportional to the amount of living cells, and the color is darker as the cells proliferate more and faster; the more cytotoxic the color is lighter. The method has higher sensitivity, better formazan dissolubility, more stability and easier preservation, and WST-8 and PMS can be prepared into a reagent which is more convenient to use. However, the kit based on the WST-8 detection still has the following drawbacks: the detection liquid containing PMS is pink with the same color as the culture medium, so that the detection liquid is easy to be added in a missing way or added in an excessive way; the method has no matched reaction termination reagent, cannot control the reaction time, and is not suitable for screening large-batch drugs; the stability of the detection liquid is poor, and the detection liquid is easy to deteriorate when being stored or transported at a higher temperature; further improvement in reaction time and detection sensitivity is required.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art and provides a cell proliferation and toxicity detection kit which can improve the detection sensitivity and stability, shorten the reaction time and avoid the leakage of detection liquid.
The technical solution of the invention is as follows: a cell proliferation and toxicity detection kit comprises detection liquid, and is characterized in that the detection liquid consists of WST-8, 2-methyl-1, 4-naphthoquinone, a sodium chloride aqueous solution, a potassium chloride aqueous solution and a calcium chloride aqueous solution, wherein the molar ratio of the WST-8 to the 2-methyl-1, 4-naphthoquinone is 12-18: 1, the concentrations of the sodium chloride aqueous solution, the potassium chloride aqueous solution and the calcium chloride aqueous solution are respectively 8.6g/L, 0.3 g/L and 0.28 g/L; and the reaction termination solution is 0.05-0.15 mol/L HCl.
Compared with the prior art, the invention has the following beneficial effects:
1. 2-methyl-1, 4-naphthoquinone is used as an electronic coupling agent, the prepared detection liquid is yellow, color change can be obviously seen after the detection liquid is added into a culture medium, the detection liquid is prevented from being added in an omission mode or in an excessive mode, and errors are reduced.
2. Compared with conventional normal saline or PBS (phosphate buffer solution), the adopted balanced salt system can provide a more stable storage environment, avoids deterioration of detection liquid due to oxidation-reduction reaction, and still cannot influence the detection effect after being placed at 50 ℃ for 20 hours. The balanced salt system of the invention is closer to the cell environment, has less influence on cells and ensures that the detection result is more accurate.
3. The reaction stopping solution is prepared, and the reaction product is added into the reaction stopping solution and then placed at 4 ℃ without crystallization, so that the absorbance can be conveniently read for many times; the detection time can be automatically controlled by the reaction stop solution, and the method is suitable for large-batch cell proliferation and toxicity detection.
4. The ratio of WST-8 to 2-methyl-1, 4-naphthoquinone is optimized, higher detection sensitivity is provided, the absorbance has a good linear relation between 0.1 and 2.8, and the detection range is wider than that of the conventional kit; after the detection solution is added for incubation for 20 minutes, the appropriate reaction degree (namely, the absorbance can be read) can be reached, the time is saved, and the efficiency is improved.
Drawings
FIG. 1 is a graph showing a standard cell number of C6 cells in example 1 of the present invention.
FIG. 2 shows the results of the cytotoxicity of paclitaxel on Hela in example 2 of the present invention.
Detailed Description
Example 1:
1. the preparation method of the cell proliferation and toxicity detection kit for 100 times of usage amount comprises the following steps:
preparation of detection liquid: 8.6 mg of sodium chloride, 0.3 mg of potassium chloride and 0.28 mg of calcium chloride are weighed and fully dissolved in 1 ml of ultrapure water, then 7.8 mg of WST-8 and 0.138 mg of 2-methyl-1, 4-naphthoquinone are added into the ultrapure water, after the complete dissolution, the mixture is filtered through a 0.22 mu m filter membrane for sterilization, and then the mixture is filled into a sterilized 1.5 ml Keli brown tube.
2. Preparation of a reaction termination solution: add 8.5. mu.l HCl into 991.5. mu.l ultrapure water, mix well, filter to sterilize with 0.22 μm filter membrane, pack in 1.5 ml transparent tube.
3. The two reagents are qualified through sterile detection and are filled into a kit according to the ratio of 1:1, and the cell proliferation and toxicity detection kit is formed.
The method for counting and detecting the activity and proliferation of the C6 cells by using the kit provided by the embodiment of the invention comprises the following steps:
1. a C6 cell suspension was prepared and the cells were counted. According to the appropriate number of plated cells (0,234,469,938,1875,3750,7500,15000,30000), approximately 100. mu.l of cell suspension per well, 3 replicate wells were set. Culturing for 2-4 hours in an incubator at 37 ℃ to make the cells adhere to the wall.
2. Add 10. mu.l of assay per well and gently tap the plate to help mix; or directly preparing a culture medium containing 10% of detection solution, and adding the culture medium in a liquid change mode;
3. incubating in an incubator for 1-2 hours, adding 10 mu l of reaction termination solution into each hole, and measuring the absorbance at 450 nm.
4. A standard curve is prepared with the number of cells as the abscissa (X-axis) and the absorbance as the ordinate (Y-axis), and the number of cells in an unknown sample can be determined from the standard curve.
Cell viability was measured under the same experimental conditions using the Japanese Dojinglen CCK-8 kit as a control, and the prepared standard curve of cell number is shown in FIG. 1. The result shows that the ratio of absorbance to cell number measured by the kit is larger, and the sensitivity is higher.
Example 2:
the preparation method of the cell proliferation and toxicity detection kit for 100 times of usage amount comprises the following steps:
1. preparation of detection liquid: weighing 8.6 mg of sodium chloride, 0.3 mg of potassium chloride and 0.28 mg of calcium chloride, fully dissolving in 1 ml of ultrapure water, adding 7.2 mg of WST-8 and 0.12 mg of 2-methyl-1, 4-naphthoquinone, sterilizing by a 0.22 mu m filter membrane after fully dissolving, and subpackaging in a sterilized 1.5 ml of Kekeishu tube.
2. Preparation of a reaction termination solution: adding 9 μ l HCl into 991 μ l ultrapure water, mixing, filtering with 0.22 μm filter membrane for sterilization, and packaging in 1.5 ml transparent tubes.
3. The two reagents are qualified through sterile detection and are filled into a kit according to the ratio of 1:1, and the cell proliferation and toxicity detection kit is formed.
The method for detecting the toxicity of paclitaxel on Hela cells by using the kit in the embodiment 2 of the invention comprises the following steps:
1. hela cell suspension was prepared and the cells were counted. According to the appropriate number of plated cells (40000), each well was approximately 100. mu.l of cell suspension, and 3 duplicate wells were set. Culturing for 2-4 hours in an incubator at 37 ℃ to make the cells adhere to the wall.
2. Each well was added with 10. mu.l of paclitaxel at various concentrations (0, 10,40,160,625,1500,2000, 2500. mu.g/ml) and incubated in an incubator at 37 ℃ for 48 hours (the incubation time of the test drug is determined according to the nature of the test drug and the sensitivity of the cells, and is generally determined according to the cell cycle, at least for one more generation).
3. Add 10. mu.l of assay per well and gently tap the plate to help mix; or directly preparing a culture medium containing 10% of detection solution, and adding the culture medium in a liquid change mode.
4. Incubating in an incubator for 1-2 hours, adding 10 mu l of reaction termination solution into each hole, and measuring the absorbance at 450 nm.
5. Calculating the survival rate and the inhibition rate of the cells:
cell viability = [ (As-Ab)/(Ac-Ab) ] × 100%;
inhibition rate = [ (Ac-As)/(Ac-Ab) ]. times.100%;
as: absorbance of experimental well (containing culture medium, detection solution, and drug)
Ac: absorbance of control well (containing culture medium, detection solution, no drug)
Ab: absorbance of blank well (cell and drug-free Medium, detection solution)
Using the Japanese Dojingzhan CCK-8 kit as a control, the toxic effect of paclitaxel on Hela cells was examined under the same experimental conditions, and a line graph was prepared with paclitaxel concentration as abscissa and absorbance as ordinate, as shown in FIG. 2. The result shows that the ratio of absorbance to drug concentration measured by the kit is larger, which indicates that the reaction to the change of the drug concentration is more sensitive.
Example 3:
the preparation method of the cell proliferation and toxicity detection kit comprises the following steps of 500 times of usage amount:
1. preparation of detection liquid: 43 mg of sodium chloride, 1.5 mg of potassium chloride and 1.4 mg of calcium chloride were weighed out and sufficiently dissolved in 5 ml of ultrapure water, 33 mg of WST-8 and 0.52 mg of 2-methyl-1, 4-naphthoquinone were added thereto, and after sufficiently dissolved, sterilized with a 0.22 μm filter, and then dispensed into 8 ml sterilized brown liquid bottles.
2. Preparation of a reaction termination solution: adding 42.5 μ l HCl into 4.96 ml ultrapure water, mixing well, filtering with 0.22 μm filter membrane for sterilization, and subpackaging in 8 ml natural color liquid bottles.
3. The two reagents are qualified through sterile detection and are filled into a kit according to the ratio of 1:1, and the cell proliferation and toxicity detection kit is formed.
The method for detecting the activity and proliferation of the suspension cells by using the kit of the embodiment 3 of the invention comprises the following steps:
1. cell suspensions were prepared and cells were counted. According to the appropriate number of plated cells, each hole of about 100 u l cell suspension, set up 3 repeat hole.
2. Add 10. mu.l of assay to each well and gently tap the plate to aid in mixing.
3. And (5) incubating in the incubator for 1-2 hours (for most adherent cells, incubating for 30 minutes).
4. Mu.l of a reaction termination solution was added to each well, and absorbance at 450nm was measured. If the absorbance is not measured temporarily, the cover culture plate can be kept at 2-8 ℃ in the dark after the reaction termination solution is added, and the absorbance does not change within 7 days.
Example 4:
the preparation method of the cell proliferation and toxicity detection kit comprises the following steps of 500 times of usage amount:
1. preparation of detection liquid: 43 mg of sodium chloride, 1.5 mg of potassium chloride and 1.4 mg of calcium chloride are weighed and fully dissolved in 5 ml of ultrapure water, then 30 mg of WST-8 and 0.52 mg of 2-methyl-1, 4-naphthoquinone are added into the ultrapure water, and after the complete dissolution, the ultrapure water is sterilized by a 0.22 mu m filter membrane and is subpackaged in 8 ml of sterilized brown liquid bottles.
2. Preparation of a reaction termination solution: adding 43 μ l HCl into 4.96 ml ultrapure water, mixing well, filtering with 0.22 μm filter membrane for sterilization, and subpackaging in 8 ml natural color liquid bottles.
3. The two reagents are qualified through sterile detection and are filled into a kit according to the ratio of 1:1, and the cell proliferation and toxicity detection kit is formed.
Method for suspension cytotoxicity detection using the kit:
1. cell suspensions were prepared and cells were counted. According to the appropriate number of plated cells, each hole of about 100 u l cell suspension, set up 3 repeat hole.
2. Adding 0-10 mul of drugs to be detected with different concentrations into each hole, and culturing for a proper time in an incubator at 37 ℃. The incubation time of the test drug is determined based on the properties of the test drug and the sensitivity of the cells, and is generally determined based on the cell cycle, at least for one or more generations.
3. Add 10. mu.l of assay to each well and gently tap the plate to aid in mixing.
4. And (5) incubating in the incubator for 1-2 hours (for most adherent cells, incubating for 30 minutes).
5. Mu.l of a reaction termination solution was added to each well, and absorbance at 450nm was measured. If the absorbance is not measured temporarily, the cover culture plate can be kept at 2-8 ℃ in the dark after the reaction termination solution is added, and the absorbance does not change within 7 days.
6. Calculating the survival rate and the inhibition rate of the cells:
cell viability = [ (As-Ab)/(Ac-Ab) ] × 100%;
inhibition rate = [ (Ac-As)/(Ac-Ab) ]. times.100%;
as: absorbance of experimental well (containing culture medium, detection solution, and drug)
Ac: absorbance of control well (containing culture medium, detection solution, no drug)
Ab: absorbance of blank wells (medium without cells and drug, test solution).
Claims (1)
1. A cell proliferation and toxicity detection kit comprises detection liquid, and is characterized in that the detection liquid consists of WST-8, 2-methyl-1, 4-naphthoquinone, a sodium chloride aqueous solution, a potassium chloride aqueous solution and a calcium chloride aqueous solution, wherein the molar ratio of the WST-8 to the 2-methyl-1, 4-naphthoquinone is 12-18: 1, the concentrations of the sodium chloride aqueous solution, the potassium chloride aqueous solution and the calcium chloride aqueous solution are respectively 8.6g/L, 0.3 g/L and 0.28 g/L; and the reaction termination solution is 0.05-0.15 mol/L HCl.
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| CN201811234278.XA CN109406765B (en) | 2018-10-23 | 2018-10-23 | Cell proliferation and toxicity detection kit |
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| CN109406765A CN109406765A (en) | 2019-03-01 |
| CN109406765B true CN109406765B (en) | 2022-02-01 |
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| US20030162164A1 (en) * | 2001-04-20 | 2003-08-28 | Biolog, Inc. | Comparative phenotype analysis of cells, including testing of biologically active compounds |
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| CN102559844A (en) * | 2012-01-09 | 2012-07-11 | 上海交通大学 | Preparation method of detecting system for minimal inhibitory concentration of mycobacteria |
| CN104955845A (en) * | 2012-09-27 | 2015-09-30 | 美国政府(由卫生和人类服务部的部长所代表) | Mesothelin antibodies and methods for eliciting potent antitumor activity |
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