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Method for evaluating adherence strength of cells by utilizing cell detachment agent

A cell and detachment technology, applied in the field of measurement, can solve the problems of high instrument and technical threshold, cumbersome operation, and difficulty in automation, and achieve the effects of improving measurement accuracy and parallelism of results, simplifying experimental steps, and reducing experimental time-consuming

Active Publication Date: 2012-10-03
北京泰盛生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to avoid the deficiencies of the prior art, the present invention proposes a method for evaluating the strength of cell attachment using a cell detachment reagent, which overcomes the shortcomings of the prior art which are cumbersome to operate, not easy to realize automation, and the threshold of instruments and techniques is too high

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: Using the method of the present invention to detect cell adhesion strength

[0022] Step 1: Prepare the human osteosarcoma cell line MG-63 at 4 x 10 4 piece / cm 2 Cells were seeded in a 96-well cell culture plate at a density of 1.4×10 4 50 wells were inoculated, and 200 μl of medium was added to each well. Place in a 37°C cell culture incubator for 24 hours to grow to a confluence of 80%;

[0023] Step 2: using trypsin solution as the cell detachment reagent, the cell detachment reagent is half-diluted to obtain a set of detachment reagent solution concentrations, and the dilution times of the concentration gradients of each detachment reagent solution are respectively 2 0 ,2 1 ,2 2 ,2 3 ,2 4 ,2 5 ,2 6 The concentration of each gradient solution is 50%, 25%, 12.5%, 6.25%, 3.13%, 1.57%, 0.79% of the standard solution concentration respectively;

[0024] Step 3: Aspirate the culture medium of 45 wells in the 96-well plate of the dry cell culture pl...

Embodiment 2

[0036] Embodiment 2: Using the method of the present invention to detect cell adhesion strength

[0037] 1. Prepare the human osteosarcoma cell line MG-63 at 4×10 4 piece / cm 2 Cells were seeded in 96-well plates at a density of 1.4×10 4 50 wells were inoculated, and 200 μl of medium was added to each well. Place in a 37°C cell culture incubator for 24 hours to grow to a confluence of 80%;

[0038] 2. Take the EDTA solution as the standard solution, dilute it twice and a half, and prepare a concentration gradient solution. The concentrations of each gradient solution are 50%, 25%, 12.5%, 6.25%, 3.13%, 1.57%, and 0.79% of the concentration of the standard solution;

[0039] 3. Drain the medium in 45 wells of the 96-well plate, and treat the cells with PBS and EDTA concentration gradient solution (0.79%-100% standard solution concentration), 100 μl per well, and make 5 replicates for each concentration. The last 5 wells with culture medium were used as the control of cell see...

Embodiment 3

[0049] Embodiment 3: Using the method of the present invention to detect cell adhesion strength

[0050] 1. Prepare GFP-labeled mouse calvarial bone cell line MC 3T3-E1 at 4×10 4 piece / cm 2 Cells were seeded in 96-well plates at a density of 1.4×10 4 50 wells were inoculated, and 200 μl of medium was added to each well. Place in a 37°C cell culture incubator for 24 hours to grow to a confluence of 80%;

[0051] 2. Take the EDTA solution as the standard solution, dilute it twice and a half, and prepare a concentration gradient solution. The concentrations of each gradient solution are 50%, 25%, 12.5%, 6.25%, 3.13%, 1.57%, and 0.79% of the concentration of the standard solution;

[0052] 3. Drain the medium in 45 wells of the 96-well plate, and treat the cells with PBS and EDTA concentration gradient solution (0.79%-100% standard solution concentration), 100 μl per well, and make 5 replicates for each concentration. The last 5 wells with culture medium were used as the contr...

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Abstract

The invention relates to a method for evaluating the adherence strength of cells by utilizing a cell detachment agent. The method is technically characterized in that the adhesive effect between the cells and a substrate is weakened by using the cell detachment agent serving as a common experimental agent in cell biology, the cells are fixed later, the number of the adherent cells of each treatedconcentration gradient treatment group is detected, a detection value of a phosphate buffer solution (PBS) treatment group is used as a standard value and normalization analysis is performed, a curvegraph which takes NA as horizontal coordinates and takes D as vertical coordinates is drawn, and the adhesion strength of the cells is characterized by dilution multiples D50 percent corresponding to1 / 2NA(PBS). Compared with the prior art, the method has the advantages that time consumed in experiments is reduced, experimental steps are simplified, automated batch treatment is easy to realize, and the measuring accuracy and the parallelity of the results are improved.

Description

technical field [0001] The invention relates to a measurement method, in particular to a method for evaluating cell adhesion strength by using a cell detachment reagent. Background technique [0002] The detection of cell adhesion strength is a problem often encountered in life science research and basic medical research. Existing methods for detecting cell adhesion strength have their own limitations: the detection method using a femtosecond laser emission device and an atomic force microscope (such as the method described in "Noncontact estimation of intercellular breaking force using a femtosecond laser impulse quantified by atomic force microscopy" ) Although the measurement is accurate and does not cause any contact damage to the cells, it is not conducive to the popularization and wide-scale application of this technology due to the high equipment and technical threshold required, the long detection time and cumbersome operation; the rotating disk method (such as Alth...

Claims

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Application Information

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IPC IPC(8): C12Q1/06
Inventor 李京宝商澎王蒙王佳李亚楠骞爱荣
Owner 北京泰盛生物科技有限公司
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