Method for testing in vitro cytotoxicity of cigarette smoke

A technology of cytotoxicity and testing methods, which is applied in the direction of biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of long cycle and cumbersome test operation steps, and achieve the effect of simple operation, flexible test time and high sensitivity

Active Publication Date: 2012-08-01
ZHENGZHOU TOBACCO RES INST OF CNTC
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Problems solved by technology

Most of the methods currently used in the evaluation of the cytotoxicity of cigarette smoke are based on the principle of cell viability measurement, but the relevant test procedures are cumbersome and the cycle is long

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  • Method for testing in vitro cytotoxicity of cigarette smoke
  • Method for testing in vitro cytotoxicity of cigarette smoke
  • Method for testing in vitro cytotoxicity of cigarette smoke

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Embodiment Construction

[0034] Evaluation of Kentucky Reference Cigarette 3R4F

[0035] After digesting the expanded and cultured CHO cells, prepare the cell suspension (1×10 5 pieces / ml). Add 100 μl of growth medium to each well of the outermost 36 wells of the 96-well plate as a blank control, add 100 μl of cell suspension to each well, and store at 37 °C, 5% CO 2 Incubate under the condition for 24 h. After the cells were cultured for 24 h, the culture medium was sucked off, and each column of 6 wells in a 96-well plate (except the outermost 36 wells) was set as a negative control, and 8 different doses (10, 50, 75, 100, 120, 140 , 160, 200 μg / ml) of total particulate matter in flue gas exposure and positive control treatment. Add 100 μl of diluted culture solution containing negative control, total particulate matter in smoke or positive control to each well, add 100 μl of diluted culture solution to each well of the outermost 36 wells of the 96-well plate as a blank control, and store at 37 ...

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Abstract

The invention discloses a method for testing in vitro cytotoxicity of cigarette smoke. The method is characterized by comprising the following steps of: 1) preparing a laboratory reagent; 2) performing cell inoculation culture; 3) performing cigarette smoke contamination; 4) dyeing with water-soluble tetrazolium-1 (WST-1); and 5) obtaining a result and analyzing. Compared with the prior art, the method is characterized in that a test step for washing cells for multiple times is eliminated in the whole test process, so that the method is easy and convenient to operate; a step of replacing a nutrient solution is not required during dyeing with the WST-1, and a diluted nutrient solution can be directly added for reacting; formazan produced by the WST-1 is water-soluble, so that a subsequent dissolution step is eliminated; and absorbance detection can be finished in 2 hours after WST-1 dyeing, so that the test period is shortened, and the method is quick in comparison with the conventional testing method. The measuring method is quick and convenient to operate, and also has the advantages of high sensitivity and stable result; and the method can be applied to smoke cytotoxicity test of multiple cell lines.

Description

[0001] technical field [0002] The invention relates to the research field of in vitro toxicological evaluation of cigarette smoke, in particular to a method for testing the in vitro cytotoxicity of cigarette smoke. [0003] Background technique [0004] The in vitro toxicological study of cigarette smoke has become one of the important means to evaluate the health hazards of smoking. At present, most of the in vitro toxicological studies on cigarette smoke focus on the cytotoxicity and genotoxicity of smoke condensate. How to scientifically and accurately evaluate the harm of low-harm cigarette products and tobacco additives, there is no uniform method and procedure in the world. In biological experimental research, the determination of cell viability is a key experimental technique. Most of the methods currently used to evaluate the cytotoxicity of cigarette smoke are based on the principle of cell viability measurement, but the relevant test procedures are cumbersome a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02
Inventor 李翔聂聪尚平平孙学辉杨松王宜鹏刘惠民
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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