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Method for measuring T-cell membrane fat micro domain STAT 5a and STAT 5b distribution and uses thereof

A cell membrane and micro-area technology, applied in biochemical equipment and methods, microbial determination/inspection, measurement devices, etc., to achieve the effect of promoting cell proliferation

Inactive Publication Date: 2005-09-07
NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Domestic studies on membrane fat microdomains and receptor signaling proteins have not been reported

Method used

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  • Method for measuring T-cell membrane fat micro domain STAT 5a and STAT 5b distribution and uses thereof
  • Method for measuring T-cell membrane fat micro domain STAT 5a and STAT 5b distribution and uses thereof
  • Method for measuring T-cell membrane fat micro domain STAT 5a and STAT 5b distribution and uses thereof

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Experimental program
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Embodiment 1

[0026] 1. Experimental materials

[0027] Human Jurkat E6-1T cells (American Type Culture Collection, Bethesda, MD), RPMI1640 medium, newborn bovine serum were from Invitrogen (Grand Island, NY); human interleukin-2 (hIL-2), protease inhibitor cocktail and BM chemiluminescent Western blotting kit was purchased from Roche Company (Indianapolis, IN); rabbit anti-human STAT5a and STAT5b antibodies were purchased from Upstate Company (Lake Placid, NY); anti-rabbit horseradish peroxidase-conjugated antibody was purchased from Roche Company ( Indianapolis, IN).

[0028] 2. Cell culture

[0029] Add RPMI 1640 medium to human Jurkat E6-1T cells, add 10% heat-inactivated newborn bovine serum (Hangzhou Sijiqing), penicillin 100U / ml medium, and streptomycin 100μg / ml to the medium. base in a carbon dioxide incubator at 5% CO 2 Cultured at 37°C. Cells were stimulated with 400 U / ml IL-2 for 30 min before cell rupture.

[0030] 3. Isolation of Membrane Fatty Microdomains

[0031] Membr...

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Abstract

The invention discloses a method of measuring the distribution of T cell membrane fats area STAT5a and STAT5b, which comprises the following steps: (a) culturing the T cell, stimulating the cell before beaching by the IL-2; (b) separating the T cell membrane fats areas through non-sequence sucrose density gradient supercentrifugation method; (c) analyzing the STAT5a and STAT5b in the separated area through protein immune brand method. The method can measure the distribution effectively and precisely, which can be used for developing the new antineoplasti and measuring the effect of it.

Description

technical field [0001] The present invention relates to a method for measuring the distribution of STAT5a and STAT5b in T cells, in particular to a method for measuring the distribution of STAT5a and STAT5b in T cell membrane fat microregions. applications in measurement. Background technique [0002] T cell membrane lipid microdomains (lipid rafts) are functional regions within the cell membrane liquid lipid bilayer (see figure 1 ), rich in glycolipids, sphingomyelin and cholesterol. The role of membrane fat microregions in some cell signal transduction has been clarified. Many important receptors and cell signaling proteins are concentrated in membrane fat microregions. The compartmentalization of receptor signaling molecules has important functional significance. Recent research results have shown that many important molecules of signal transduction pathways, such as T cell receptor (TCR / CD3), B cell receptor (BCR), and IgE receptor, exist in the lipid rafts region. T ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/00G01N33/53G01N33/574G01N33/68
Inventor 李秋荣黎介寿
Owner NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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