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In-vitro cell micronuclei method for detecting total particulate matters of main stream smoke of cigarettes

A technology for mainstream smoke and particulate matter of cigarettes, which is applied in the preparation of test samples, measuring devices, and individual particle analysis, etc., to achieve the effects of less cell volume, simple operation and large sample volume

Active Publication Date: 2014-10-29
CHINA TOBACCO YUNNAN IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no study on the application of high-content imaging system to the detection of micronucleus in vitro for total particulate matter in mainstream cigarette smoke

Method used

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  • In-vitro cell micronuclei method for detecting total particulate matters of main stream smoke of cigarettes
  • In-vitro cell micronuclei method for detecting total particulate matters of main stream smoke of cigarettes
  • In-vitro cell micronuclei method for detecting total particulate matters of main stream smoke of cigarettes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] In vitro cell micronucleus test of a high-content imaging system for total particulate matter (TPM) in mainstream smoke of a domestic cigarette.

[0034] Follow the above steps (1) to prepare the mainstream smoke total particulate matter (TPM) of cigarettes, then follow the steps (2) for cell culture, and then set 25, 50, 100, 150, 200 μg / ml of TPM to infect the cells , Cytochalasin B with a final concentration of 6 μg / ml was added to all groups, and the volume concentration was 5% CO 2 , cultured at 37°C for 24 hours, then fixed the cells according to step (4), permeabilized the cells according to step (5), stained the cell nucleus according to steps (6) and (7), and then used high-content imaging The system detects, selects FITC and DAPI fluorescence dual channels to scan the cells to obtain pictures, and uses HCS micronucleus analysis software to analyze the pictures after scanning, and sets the main nucleus and micronuclei according to the difference between the flu...

Embodiment 2

[0038] Example 2 In vitro cell micronucleus test of a high-content imaging system for mainstream smoke total particulate matter (TPM) of a certain domestic cigarette. The specific embodiment refers to embodiment 1. The frequency of micronuclei induced by different concentrations of TPM in a domestic cigarette detected by the high-content imaging system (HCS) is shown in Table 2.

[0039] Table 2. The rate of micronuclei induced by different concentrations of TPM in a domestic cigarette detected by the high-content imaging system (‰)

[0040]

[0041] It can be known from Table 2 that as the concentration of TPM increases, the rate of micronuclei induced by it increases, showing a significant dose-response relationship.

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Abstract

The invention discloses an in-vitro cell micronuclei method for detecting total particulate matters of main stream smoke of cigarettes. The method comprises the steps of seeding cells on a 96-hole plate for cigarette smoke contamination, directly fixing, dyeing, and feeding the cells into a high-connotation imaging system for detection; setting fluorescence intensity parameters of main nucleuses and the micronuclei of the cells according to the difference between a fluorescence value of cell nucleuses and a background fluorescence value, measuring the sizes of the main nucleuses and the micronuclei of the cells so as to set nucleus diameter parameters, and setting distance parameters according to distances between the cell nucleuses; performing analysis on images according to the setting, and selecting the number of binucleated cells with the micronuclei and the total number of the binucleated cells from a result lead-out column so as to calculate the micronucleus rate. According to the method, the number of required cells is small, the cells are not need to be used, and micronuclei sheets are not required to be manufactured; furthermore, the in-vitro cell micronuclei method is high in detection speed, large in sampling quantity and high in accuracy.

Description

technical field [0001] The invention relates to an in vitro cell micronucleus method for detecting total particulate matter (TPM) in cigarette mainstream smoke by using a high-content imaging system, and belongs to the technical field of cigarette smoke safety evaluation. Background technique [0002] The in vitro cell micronucleus test is one of the in vitro toxicological tests currently used for the safety assessment of cigarette smoke, and is used to evaluate the genotoxicity of cigarette smoke. The traditional micronucleus test is to directly count the micronucleus rate of the material to be tested under a microscope after microscopic preparation using cell biology methods. The traditional micronucleus detection method requires a large amount of cells, and the cells need to be digested before micronucleus production, so that the loss of cells is large, the damage is large, and the later stage is manually read, and the detection sample size is limited and the accuracy is ...

Claims

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Application Information

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IPC IPC(8): G01N15/10G01N1/30G06T7/00
Inventor 高茜缪明明夭建华曾婉俐朱洲海管莹米其利李雪梅
Owner CHINA TOBACCO YUNNAN IND
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