Method of low temperature induction off-spreading of cell for dynamic measurement of medicament influence on cell spreading

A low-temperature induced, dynamic measurement technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of complex research and analysis, protein damage, etc., and achieve the effect of rapid detection and simple process

Inactive Publication Date: 2012-06-27
NANCHANG UNIV
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  • Summary
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  • Application Information

AI Technical Summary

Problems solved by technology

However, trypsin has a greater damage effect on various proteins on the surface of the cell membrane, and it is not clear whether it will affect various physiological activities of cells (such as cell spreading, etc.). Therefore, this biochemical method will make the drug affect The research and analysis of cell spreading is more complicated, and there is an urgent need for a more gentle, side-effect-free, and effective method of cell spreading

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Quantitative measurement of low temperature induced cell despreading.

[0025] The suspended human umbilical vein endothelial cells were planted on a 24-well plate containing culture medium and placed in CO 2 Incubator (37°C, 5% CO 2 ) cultured. After 24 hours of culture, the cells were placed in the cell culture system that came with the confocal microscope (37°C, 5% CO 2 ), observe with a confocal microscope and acquire a cell topography image of a field of view. Add culture medium at a temperature of 4°C, and immediately use the dynamic observation software that comes with the confocal microscope to automatically scan the cell morphology image of the same field of view every 1 minute. Use the measurement software that comes with the microscope to measure the adherent area of ​​all single cells in the images at each time point and calculate the average value. Only the data for each 5-minute period are given here. Calculated: the average spreading area ...

Embodiment 2

[0026] Example 2: Low temperature induces cell despreading Quantitative measurements for measuring the effect of drugs on cell spreading.

[0027] The experimental procedure is basically the same as that of Experimental Example 1, except that the drug (10ug / ml ganglioside GM1) was added after the cells were cultured for 24 hours, and the drug was removed after co-incubating with the cells for 30 minutes and replaced with fresh culture medium, and the sample was placed Cells were induced to despread under a microscope at low temperature and dynamically observed and measured. Calculated: the average spread area of ​​drug-treated cells before low-temperature treatment was 1053.9±396.8 um 2 ; 1 minute after low temperature treatment, the area is 983.8±350.9 um 2 ; Thereafter, every 5 minutes were 933.0±298.4 um 2 (5 minutes), 702.9±266.9 um 2 (10 minutes), 640.7±240.5 um 2 (15 minutes), 605.5±221.1 um 2 (20 minutes), 593.5±213.6 um 2 (25 minutes), 643.9±240.3 um 2 (30th min...

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Abstract

A method of low temperature induction off-spreading of cell for dynamic measurement of medicament influence on cell spreading The method is characterized by comprising the following steps: planting adherent cells in a suspension state on a culture aperture or plate and incubating in an incubator to complete spread the cells; adding medicament and incubating the cells and the medicament together for 20 min-1 h and removing the medicament; placing the cells treated by the medicament under a microscope and shooting a morphology image before cell spreading; absorbing the aqueous solution and adding 4 DEG C cell nutrient solution or buffer; dynamically observing off-spreading state of the cells by a microscope immediately, continuously obtaining morphology images of the cells every few minutes for 30 min-1 h; measuring adherence area of each cell or a mean value of adherence areas of all the cells at a certain time point by a piece of software; and comparing the obtained data with blank data and analyzing to obtain data of medicament influence on cell spreading. The invention has advantages of simple process, no damage on cells, rapid detection, dynamic observation or measurement and good effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and is a method for measuring the influence of various drugs on the spreading state of adherent cells. technical background [0002] Cell spreading is one of the important physiological activities or functions of adherent cells, and many human physiological activities or diseases are related to cell spreading. For example, vascular endothelial cells build the inner wall of blood vessels or repair the damaged vascular endothelial layer through cell spreading. Therefore, changes in endothelial cell spreading are often related to the occurrence, development and tumor metastasis of cardiovascular diseases, and the effect of drugs on cell spreading It must also be one of the important indicators in in vitro cell tests to evaluate the efficacy of drugs. [0003] Currently, there are two methods, direct and indirect, to measure the effect of drugs on cell spreading state. The former is to add the drug dir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02
Inventor 陈勇邵文祥黄洁曾芳发
Owner NANCHANG UNIV
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