Method for separating Pig-a gene mutant cells from cells cultured in vitro

An in vitro culture and cell technology, applied in the field of biological cells, can solve the problems of high cost and low efficiency, and achieve the effects of low cost, simple operation and high separation efficiency

Pending Publication Date: 2020-03-17
SHANGHAI INNOSTAR BIO TECH
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  • Abstract
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Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a met

Method used

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Experimental program
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Embodiment 1

[0035] The separation of embodiment 1 Pig-a gene mutation cell

[0036] 1. After the logarithmic phase cell count, take 6 million cells, divide into three parts, centrifuge at 1000RPM for 5min, discard the supernatant, add 10mL 2-8℃ pre-cooled PBS, and wash once. Centrifuge again to discard the supernatant, leaving about 10 μL of liquid in each tube to resuspend the cells.

[0037] 2. Add 175 μL of antibody staining solution to a 1.5mL conical centrifuge tube, gently add the cell suspension to the antibody staining solution, and mix gently to make all cells contact with the antibody, preventing cells from being unlabeled to the antibody and causing false positives. Positive. Under dark conditions, incubate on ice for 30±5min. Add 2 mL of pre-cooled PBS containing 2% BSA, centrifuge and discard the supernatant, leaving about 10 μL of liquid in each tube to resuspend the cells.

[0038]3. Add 150 μL of anti-PE-magnetic beads to a 1.5 mL conical centrifuge tube, gently add the...

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Abstract

The invention discloses a method for separating Pig-a gene mutant cells from cells cultured in vitro and application of the Pig-a gene mutant cells. The method comprises the following steps: (1) mixing an anti-GPI anchorage protein antibody marked by an immunofluorescent dye, magnetic beads resisting the immunofluorescent dye and the cells; and (2) separating cells marked by the magnetic beads from cells not marked by the magnetic beads, wherein the cells marked by the magnetic beads being Pig-a gene mutation negative cells, and the cells not marked by the magnetic beads being Pig-a gene mutation positive cells. The method provided by the invention has higher separation efficiency for removing cells, such as the Pig-a positive cells pre-stored in human lymphoblastic TK6 cells, can separate10 million cells within 15 minutes, and is simple to operate and low in cost.

Description

technical field [0001] The invention belongs to the field of biological cells, in particular to a method for isolating Pig-a gene mutant cells from cells cultured in vitro. Background technique [0002] The Pig-a gene is located on the short arm of the human X chromosome and is involved in the early synthesis of glycosylphosphatidylinositol (GPI) linker proteins. In the genetic toxicology detection test, the Pig-a gene mutation test can be used to evaluate the ability of the compound to induce gene mutation. The Pig-a gene mutation test uses flow cytometry (flowcytometry, FCM) to detect the expression of GPI-anchored proteins (such as CD59, CD55 protein) on the cell surface, and evaluates the gene induced by the test substance through the change of the Pig-a gene mutation rate. ability to mutate. [0003] The existing Pig-a gene mutation method is mainly the animal gene mutation test using rats as the test model. However, the Pig-a gene mutation method of animal cells cul...

Claims

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Application Information

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IPC IPC(8): C12N5/00
CPCC12N5/0081C12N2509/00
Inventor 李若婉黄鹏程周长慧常艳
Owner SHANGHAI INNOSTAR BIO TECH
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