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Method and kit for measuring cell activity and application of kit

Inactive Publication Date: 2014-05-21
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the problem of cross-reaction between the detection reagent and the reducing agent in the detection system in the existing cell viability assay methods (such as MTT method

Method used

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  • Method and kit for measuring cell activity and application of kit
  • Method and kit for measuring cell activity and application of kit
  • Method and kit for measuring cell activity and application of kit

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0041] Example 1. Using 2,3,5-triphenyltetrazolium chloride (TTC, molecular formula C 19 H 15 ClN 4 ) Method for determining cell viability and determination of the optimal concentration of TTC detection solution

[0042] Technical points of this embodiment:

[0043] a) Preparation and use of TTC detection solution;

[0044] b) The absorption spectrum of formazan and its solution generated by the dissolution of dimethyl sulfoxide (DMSO);

[0045] c) The optimal concentration of TTC to determine cell viability.

[0046] This embodiment determines that 2,3,5-triphenyltetrazolium chloride (TTC, molecular formula C 19 H 15 ClN 4 ) Whether it can be used to determine cell viability and concentration, perform the following operations:

[0047] 1) Prepare TTC detection solution: use 0.01M phosphate buffer (PBS, formula: 137mM NaCl, 2.7mM KCl, 10mM Na 2 HPO 4 , 2mM KH 2 PO 4 , PH 7.2-7.4) Dissolve TTC drugs, and prepare TTC detection solutions with mass percentage concentrations of 0.1%, 0.2%, 0...

Example Embodiment

[0053] Example 2. The relationship between the absorbance value obtained by TTC measuring cell viability and the number of cells

[0054] Technical points of this embodiment:

[0055] a) The number of cells suitable for determining cell viability by TTC is 4×10 4 -2×10 5 Within range

[0056] b) Make a scatter plot and fit the curve to analyze the functional relationship between the absorbance value obtained by the TTC method and the number of cells.

[0057] This embodiment determines that 2,3,5-triphenyltetrazolium chloride (TTC, molecular formula C 19 H 15 ClN 4 ) To determine the relationship between the absorbance value obtained from cell viability and the number of cells, perform the following operations:

[0058] 1) Prepare TTC detection solution: use 0.01M phosphate buffer (PBS, formula: 137mM NaCl, 2.7mM KCl, 10mM Na 2 HPO 4 , 2mM KH 2 PO 4 , PH 7.2-7.4) Dissolve the TTC medicine and prepare a TTC detection solution with a concentration of 1.0% by mass. Use a filter with a por...

Example Embodiment

[0063] Example 3. Using TTC to measure cell viability

[0064] 1. Sample to be tested 1: Treatment of PC12 cells with drug hydrogen peroxide solution

[0065] This embodiment uses 2,3,5-triphenyltetrazolium chloride (TTC, molecular formula C 19 H 15 ClN 4 ) Determine the viability of PC12 cells in the sample. The operation process is as follows:

[0066] 1) Prepare TTC detection solution: use 0.01M phosphate buffer (PBS, formula: 137mM NaCl, 2.7mM KCl, 10mM Na 2 HPO 4 , 2mM KH 2 PO 4 , PH 7.2-7.4) dissolve TTC drugs, and prepare a TTC detection solution with a mass percentage concentration of 1.0% (0.5-1.0% is acceptable). Use a filter with a pore size of 0.22μm to filter and sterilize, separate and store at -20°C.

[0067] 2) After the PC12 cells are digested, they are inoculated into a 24-well plate and used high-glycemic DMEM medium containing 5% fetal bovine serum, 5% horse serum, 100U / mL penicillin, and 100U / mL streptomycin (for the recipe, see Life Technologies’ GIBCO 12800-01...

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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 [0001] The invention belongs to a method for measuring cell viability in the field of biotechnology, in particular to a method for measuring cell viability using 2,3,5-triphenyltetrazolium chloride (TTC). The viability of suspension culture cells is better than that of existing 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT), 3,3'-[1- (Phenyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)sodium benzenesulfonate (XTT) and water-soluble tetrazolium (WSTs), etc. for the determination of cell viability The obtained results are more reliable and are not easily interfered by antioxidants in the detection system, so that more scientific, objective and reliable results of cell viability can be obtained. Background technique [0002] In biological experiments, in order to determine the effects of certain stimuli or drugs on cell survival, proliferation, growth inhibition or toxicity, etc., it is often necessary to measure the viability of cells. Existing ...

Claims

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Application Information

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IPC IPC(8): G01N21/31G01N15/10
Inventor 张成岗何国维李伟光高艳李志慧高大文
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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