CN109406765A - Cell Proliferation and toxicity detection kit - Google Patents
Cell Proliferation and toxicity detection kit Download PDFInfo
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- CN109406765A CN109406765A CN201811234278.XA CN201811234278A CN109406765A CN 109406765 A CN109406765 A CN 109406765A CN 201811234278 A CN201811234278 A CN 201811234278A CN 109406765 A CN109406765 A CN 109406765A
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- 238000001514 detection method Methods 0.000 title claims abstract description 55
- 230000004663 cell proliferation Effects 0.000 title claims abstract description 21
- 231100000419 toxicity Toxicity 0.000 title claims abstract description 19
- 230000007541 cellular toxicity Effects 0.000 title claims abstract description 13
- 239000007788 liquid Substances 0.000 claims abstract description 58
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims abstract description 20
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 235000002639 sodium chloride Nutrition 0.000 claims abstract description 13
- VSIVTUIKYVGDCX-UHFFFAOYSA-M sodium;4-[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate Chemical compound [Na+].COC1=CC([N+]([O-])=O)=CC=C1[N+]1=NC(C=2C(=CC(=CC=2)S([O-])(=O)=O)S([O-])(=O)=O)=NN1C1=CC=C([N+]([O-])=O)C=C1 VSIVTUIKYVGDCX-UHFFFAOYSA-M 0.000 claims abstract description 11
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 10
- 239000001110 calcium chloride Substances 0.000 claims abstract description 10
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 10
- 239000001103 potassium chloride Substances 0.000 claims abstract description 10
- 235000011164 potassium chloride Nutrition 0.000 claims abstract description 10
- 239000011780 sodium chloride Substances 0.000 claims abstract description 10
- 229960002668 sodium chloride Drugs 0.000 claims abstract description 10
- FRASJONUBLZVQX-UHFFFAOYSA-N 1,4-naphthoquinone Chemical class C1=CC=C2C(=O)C=CC(=O)C2=C1 FRASJONUBLZVQX-UHFFFAOYSA-N 0.000 claims abstract 2
- RRHGJUQNOFWUDK-UHFFFAOYSA-N Isoprene Chemical compound CC(=C)C=C RRHGJUQNOFWUDK-UHFFFAOYSA-N 0.000 claims abstract 2
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 230000035484 reaction time Effects 0.000 abstract description 5
- 150000003716 vitamin K3 derivatives Chemical class 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 44
- 238000002835 absorbance Methods 0.000 description 21
- 239000003814 drug Substances 0.000 description 16
- 229940079593 drug Drugs 0.000 description 15
- 238000000034 method Methods 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 12
- 239000001963 growth medium Substances 0.000 description 10
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 8
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 8
- 239000006285 cell suspension Substances 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 230000001988 toxicity Effects 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000004090 dissolution Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- JUJBNYBVVQSIOU-UHFFFAOYSA-M sodium;4-[2-(4-iodophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate Chemical compound [Na+].C1=CC([N+](=O)[O-])=CC=C1N1[N+](C=2C=CC(I)=CC=2)=NC(C=2C(=CC(=CC=2)S([O-])(=O)=O)S([O-])(=O)=O)=N1 JUJBNYBVVQSIOU-UHFFFAOYSA-M 0.000 description 5
- 229930012538 Paclitaxel Natural products 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 229960001592 paclitaxel Drugs 0.000 description 4
- 238000012797 qualification Methods 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 238000013190 sterility testing Methods 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 4
- 239000003643 water by type Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 230000008859 change Effects 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 108010087230 Sincalide Proteins 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 230000022131 cell cycle Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 210000003470 mitochondria Anatomy 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000004904 shortening Methods 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- -1 tetramethyl azo azoles salt Chemical class 0.000 description 2
- 230000006820 DNA synthesis Effects 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 235000016408 Podocarpus macrophyllus Nutrition 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 102000019259 Succinate Dehydrogenase Human genes 0.000 description 1
- 108010012901 Succinate Dehydrogenase Proteins 0.000 description 1
- 244000162450 Taxus cuspidata Species 0.000 description 1
- 235000009065 Taxus cuspidata Nutrition 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000007259 addition reaction Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
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
- G01N33/52—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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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a kind of cell Proliferation and toxicity detection kit that detection sensitivity and stability can be improved, shorten the reaction time, avoid leakage plus detection liquid, there is detection liquid, the detection liquid is by WST-8,2- methyl-1,4- naphthoquinones, sodium-chloride water solution, potassium chloride solution and calcium chloride water group at, the WST-8 and 2- methyl-1, the molar ratio of 4- naphthoquinones is 12 ~ 18:1, and the concentration of the sodium-chloride water solution, potassium chloride solution and calcium chloride water is respectively 8.6g/L, 0.3 g/L, 0.28g/L；There is reaction terminating liquid, the reaction terminating liquid is 0.05 ~ 0.15 mol/L HCl.
Description
Technical field
The present invention relates to technical field of cell biology, especially one kind, and detection sensitivity and stability, shortening can be improved
Reaction time, the cell Proliferation and toxicity detection kit for avoiding leakage plus detection liquid.
Background technique
Cell Proliferation and toxicity detection are the basic skills for measuring substance toxicity, assessing drug safety and cell health, together
When can indicate proliferative activity o f tumor, target gene wink turn/surely turn the cell-proliferation activity of cell line, target gene crosses table
Reach or RNAi interference gene functional research, play a significant role in drug screening and functional safety Journal of Sex Research.Therefore, cell
Proliferation is widely used to molecular biology, oncobiology, immunology, science of heredity, pharmacology and medicine generation with toxicity detection technology
Each research field such as dynamics.
Currently, cell Proliferation detection method mainly includes that DNA synthesis detection, the detection of cell Proliferation related antigen and metabolism are lived
Property detection three categories, metabolic activity detection method is the most commonly used method.The metabolic activity detection method of early stage includes MTT
Method, XTT method, MTS method and WST-1 method etc. detect liquid containing MTT, XTT, MTS and WST-1 respectively that is, in detection kit.MTT
The principle of (tetramethyl azo azoles salt) method is that the succinate dehydrogenase in living cells mitochondria can make exogenous MTT be reduced to water not
The bluish violet Jie Jing formazan of dissolubility is simultaneously deposited in cell (and dead cell is without this function), after crystallization dissolution, measures 570nm wave
Long absorption value, within the scope of certain cell number, MTT crystallizes that the amount to be formed is directly proportional to cell number, and absorbance is bigger, indicates cell
Activity is stronger, illustrates that drug toxicity is smaller.Though mtt assay have many advantages, such as it is easy to operate, sensitive, "dead", due to
Rong Xie formazan product is needed, causes result error due to formazan dissolution is not complete or takes out of sometimes, therefore poor repeatability, and have
There is cytotoxicity.
XTT(tetranitroazole derivative) method is similar to MTT principle, and through living cells mitochondria dehydrogenation enzyme effect, XTT is reduced into
Water-soluble brown color formazan, in the presence of electron pair mixture sulfuric acid phenol piperazine formicester (PMS), the production quantity and work of brown color formazan
Cell quantity is positively correlated.But it since XTT itself is not readily dissolved and solution is unstable, limits its application.
MTS method and WST-1 method are similar to XTT principle, they need PMS to exist, but the shortcomings that overcome MTT and XTT,
With higher sensitivity and specificity, but MTS or WST-1 needs separately formulated with PMS, and when use mixes in proportion again,
Increase the complexity of operation.
WST-8[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-
Disulfophenyl) -2H-tetrazolium] it is a kind of water-soluble tetrazolium updated compared with WST-1 developed in recent years,
It is similar to preceding method using principle, it, can be by Intramitochondrial dehydrogenase under the conditions of electronics coupled agent (such as PMS) is existing
It is reduced to the orange-yellow formazan product with high water soluble, generation formazan quantity is directly proportional to the quantity of living cells, cell
Much faster then color is deeper for proliferation；The more big then color of cytotoxicity is more shallow.This higher , formazan dissolubility of method sensitivity is more
Good, more stable, more easy to maintain, and WST-8 and PMS can be configured to a kind of reagent, when use, is more convenient.But it is examined based on WST-8
The kit of survey still has following defects that the detection liquid containing PMS is pink colour identical with culture medium color, be easy to cause inspection
It surveys liquid leakage plus or adds；There is no matched reaction terminating reagent, be unable to control the reaction time, is not suitable for doing large batch of drug sieve
Choosing；The stability for detecting liquid is poor, and higher temperature is perishable when saving or transporting；Reaction time and detection sensitivity are needed into one
Step improves.
Summary of the invention
The present invention is to solve above-mentioned technical problem present in the prior art, and providing one kind can be improved detection sensitivity
And stability, shortening reaction time, the cell Proliferation and toxicity detection kit for avoiding leakage plus detection liquid.
The technical solution of the invention is as follows: a kind of cell Proliferation and toxicity detection kit have detection liquid, feature exists
In the detection liquid by WST-8,2- methyl-1,4-naphthaquinone, sodium-chloride water solution, potassium chloride solution and calcium chloride water group
At the molar ratio of the WST-8 and 2-MNQ is 12 ~ 18:1, the sodium-chloride water solution, potassium chloride solution
And the concentration of calcium chloride water is respectively 8.6g/L, 0.3 g/L, 0.28g/L；There are reaction terminating liquid, the reaction terminating liquid
For 0.05 ~ 0.15 mol/L HCl.
Compared with prior art, the present invention having the following beneficial effects:
1. being used as electronics coupled agent using 2-MNQ, made detection liquid is yellow, can be with after addition culture medium
It is clearly visible color change, avoid leakage plus adds to detect liquid, reduction error.
2. the balance salt system used is capable of providing more stable preservation compared with conventional physiological saline or PBS
Environment avoids detection liquid from going bad because redox reaction occurs, and places at 50 DEG C and still will not influence within 20 hours detection effect
Fruit.Balance salt system of the invention is smaller to impact cell closer to cellular environment, keeps testing result more acurrate.
3. being furnished with reaction terminating liquid, being put into 4 DEG C after reaction product addition reaction terminating liquid will not be crystallized, convenient repeatedly to read
Take absorbance；Detection time can be voluntarily controlled by reaction terminating liquid, be suitable for high-volume cell Proliferation and toxicity detection.
4. optimizing the proportion of WST-8 and 2-MNQ, higher detection sensitivity is provided, absorbance exists
All there is good linear relationship, detection range is more wider than previous kit between 0.1 ~ 2.8；20 points are added after detection liquid is incubated for
Clock can reach the suitable extent of reaction (absorbance can be read), saves the time, improves efficiency.
Detailed description of the invention
Fig. 1 is the cell number canonical plotting of C6 cell in the embodiment of the present invention 1.
Fig. 2 is taxol in the embodiment of the present invention 2 to Hela cytotoxicity testing result.
Specific embodiment
Embodiment 1:
1. the preparation method of cell Proliferation and 100 usage amounts of toxicity detection kit:
Detect the preparation of liquid: weighing 8.6 mg sodium chloride, 0.3 mg potassium chloride, 0.28 mg calcium chloride, to be completely dissolved in 1 ml super
In pure water, 7.8 mg WST-8 and 0.138 mg 2-MNQ are then added thereto, cross 0.22 μ after completely dissolution
M filter membrane degerming, 1.5 ml loaded on sterilizing can be stood in brown pipe.
2. the preparation of reaction terminating liquid: 8.5 μ l HCl being added in 991.5 μ l ultrapure waters, 0.22 μm is crossed after mixing
Filter membrane degerming can be stood in transparent pipe loaded on 1.5 ml.
3. above two reagent forms cell Proliferation and toxicity according to 1:1 loaded in kit through Sterility testing qualification
Detection kit.
The method for carrying out C6 cell count using kit of the embodiment of the present invention, detecting cell activity and proliferation:
1. preparing C6 cell suspension, cell count.According to suitable bed board cell number (0,234,469,938,1875,3750,
7500,15000,30000), about 100 μ l cell suspension of every hole, is arranged 3 repeating holes.It is cultivated 2 ~ 4 hours in 37 DEG C of incubators,
Keep cell adherent.
2. every hole is added 10 μ l and detects liquid, culture plate is tapped gently to help to mix；Or directly configuration is containing 10% detection
The culture medium of liquid is added in the form of changing liquid；
3. being incubated for 1 ~ 2 hour in incubator, the reaction terminating liquid of 10 μ l is added in every hole, measures 450nm absorbance.
4. producing one using cell quantity as abscissa (X-axis), absorbance is the standard curve of ordinate (Y-axis), root
Standard curve can determine the cell quantity of unknown sample accordingly.
Using Japanese colleague CCK-8 kit as compareing, under identical experiment condition, cell viability, production are detected
Cell number standard curve it is as shown in Figure 1.The result shows that absorbance/cell number ratio that kit of the present invention measures is bigger,
Sensitivity is higher.
Embodiment 2:
The preparation method of cell Proliferation and 100 usage amounts of toxicity detection kit:
1. detecting the preparation of liquid: weighing 8.6 mg sodium chloride, 0.3 mg potassium chloride, 0.28 mg calcium chloride and be completely dissolved in 1 ml
In ultrapure water, 7.2 mg WST-8 and 0.12 mg 2-MNQ are then added thereto, cross 0.22 after completely dissolution
μm filter membrane degerming, 1.5 ml for being sub-packed in sterilizing can be stood in brown pipe.
2. the preparation of reaction terminating liquid: 9 μ l HCl being added in 991 μ l ultrapure waters, 0.22 μm of filter membrane is crossed after mixing
Degerming, being sub-packed in 1.5 ml can stand in transparent pipe.
3. above two reagent forms cell Proliferation and poison according to 1:1 loaded in kit through Sterility testing qualification
Property detection kit.
Taxol is carried out to the detection method of toxicity of Hela cell using 2 kit of the embodiment of the present invention:
1. preparing Hela cell suspension, cell count.According to suitable bed board cell number (40000), about 100 μ l cell of every hole
3 repeating holes are arranged in suspension.It is cultivated 2 ~ 4 hours in 37 DEG C of incubators, keeps cell adherent.
2. the Japanese yew that 10 μ l various concentrations (0,10,40,160,625,1500,2000,2500 μ g/ml) is added in every hole
Alcohol is cultivated 48 hours in 37 DEG C of incubators and (determines the culture of drug to be measured according to the sensibility of the property of drug to be measured and cell
Time generally determines according to the cell cycle, at least cultivates the time of a generation or more).
3. every hole is added 10 μ l and detects liquid, culture plate is tapped gently to help to mix；Or directly configuration is containing 10% detection
The culture medium of liquid is added in the form of changing liquid.
4. being incubated for 1 ~ 2 hour in incubator, the reaction terminating liquid of 10 μ l is added in every hole, measures 450nm absorbance.
5. cell survival rate and inhibiting rate calculate:
Cell survival rate=[(As-Ab)/(Ac-Ab)] × 100%；
Inhibiting rate=[(Ac-As)/(Ac-Ab)] × 100%；
As: the absorbance of experimental port (containing culture medium, detection liquid, drug)
Ac: the absorbance of control wells (containing culture medium, detecting liquid, without drug)
Ab: the absorbance of blank well (culture medium, detection liquid without cell and drug)
Using Japanese colleague CCK-8 kit as compareing, under identical experiment condition, taxol is detected to Hela cell
Toxic effect is that ordinate production line chart is as shown in Figure 2 using paclitaxel concentration as abscissa, absorbance.The result shows that this hair
The absorbance that bright kit measures/drug concentration ratio is bigger, illustrates that the reaction changed to drug concentration is sensitiveer.
Embodiment 3:
The preparation method of cell Proliferation and 500 usage amounts of toxicity detection kit:
1. detecting the preparation of liquid: weighing 43 mg sodium chloride, 1.5 mg potassium chloride, 1.4 mg calcium chloride, to be completely dissolved in 5 ml super
In pure water, 33 mg WST-8 and 0.52 mg 2-MNQ are then added thereto, cross 0.22 μm after completely dissolution
Filter membrane degerming is sub-packed in 8 ml brown liquid bottles of sterilizing.
2. the preparation of reaction terminating liquid: 42.5 μ l HCl being added in 4.96 ml ultrapure waters, 0.22 μm is crossed after mixing
Filter membrane degerming is sub-packed in 8 ml true qualities liquid bottles.
3. above two reagent forms cell Proliferation and toxicity according to 1:1 loaded in kit through Sterility testing qualification
Detection kit.
The method for carrying out suspension cell activity and proliferation detection using 3 kit of the embodiment of the present invention:
1. preparing cell suspension, cell count.According to suitable bed board cell number, about 100 μ l cell suspension of every hole is arranged 3
Repeating hole.
2. every hole is added 10 μ l and detects liquid, culture plate is tapped gently to help to mix.
3. being incubated in incubator 1 ~ 2 hour (being incubated for 30 minutes for most of attached cells).
4. the reaction terminating liquid of 10 μ l is added in every hole, 450nm absorbance is measured.If temporarily not measuring absorbance,
It can cover culture plate after reaction terminating liquid is added and be kept in dark place at 2 ~ 8 DEG C, not change in 7 days internal absorbances.
Embodiment 4:
The preparation method of cell Proliferation and 500 usage amounts of toxicity detection kit:
1. detecting the preparation of liquid: weighing 43 mg sodium chloride, 1.5 mg potassium chloride, 1.4 mg calcium chloride, to be completely dissolved in 5 ml super
In pure water, 30 mg WST-8 and 0.52 mg 2-MNQ are then added thereto, cross 0.22 μm after completely dissolution
Filter membrane degerming is sub-packed in 8 ml brown liquid bottles of sterilizing.
2. the preparation of reaction terminating liquid: 43 μ l HCl being added in 4.96 ml ultrapure waters, 0.22 μm of filter is crossed after mixing
Film degerming is sub-packed in 8 ml true qualities liquid bottles.
3. above two reagent forms cell Proliferation and toxicity according to 1:1 loaded in kit through Sterility testing qualification
Detection kit.
The method for carrying out suspension cell toxicity detection using kit:
1. preparing cell suspension, cell count.According to suitable bed board cell number, about 100 μ l cell suspension of every hole, setting 3
A repeating hole.
2. the drug to be measured of 0 ~ 10 μ l various concentration is added in every hole, appropriate time is cultivated in 37 DEG C of incubators.According to
The sensibility of the property and cell of surveying drug determines the incubation time of drug to be measured, is generally determined according to the cell cycle, at least
Cultivate the time of a generation or more.
3. every hole is added 10 μ l and detects liquid, culture plate is tapped gently to help to mix.
4. being incubated in incubator 1 ~ 2 hour (being incubated for 30 minutes for most of attached cells).
5. the reaction terminating liquid of 10 μ l is added in every hole, 450nm absorbance is measured.If not measuring extinction temporarily
Degree can cover culture plate and be kept in dark place at 2 ~ 8 DEG C, not change in 7 days internal absorbances after reaction terminating liquid is added.
6. cell survival rate and inhibiting rate calculate:
Cell survival rate=[(As-Ab)/(Ac-Ab)] × 100%；
Inhibiting rate=[(Ac-As)/(Ac-Ab)] × 100%；
As: the absorbance of experimental port (containing culture medium, detection liquid, drug)
Ac: the absorbance of control wells (containing culture medium, detecting liquid, without drug)
Ab: the absorbance of blank well (culture medium, detection liquid without cell and drug).
Claims (1)
1. a kind of cell Proliferation and toxicity detection kit, have detection liquid, it is characterised in that the detection liquid is by WST-8,2- first
Base -1,4-naphthoquinone, sodium-chloride water solution, potassium chloride solution and calcium chloride water group at, the WST-8 and 2- methyl-1,
The molar ratio of 4- naphthoquinones is 12 ~ 18:1, and the concentration of the sodium-chloride water solution, potassium chloride solution and calcium chloride water is distinguished
For 8.6g/L, 0.3 g/L, 0.28g/L；There is reaction terminating liquid, the reaction terminating liquid 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 true CN109406765A (en) | 2019-03-01 |
| CN109406765B CN109406765B (en) | 2022-02-01 |
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