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Method for quantitatively separating and detecting infiltrative immune cells of small intestine muscularis

A detection method and immune cell technology, applied in the field of immune cell separation and detection, can solve problems such as missing cell groups

Active Publication Date: 2021-02-09
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Those skilled in the art can often only use the reported antibody combinations to type immune cells, but doing so may miss certain cell populations

Method used

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  • Method for quantitatively separating and detecting infiltrative immune cells of small intestine muscularis
  • Method for quantitatively separating and detecting infiltrative immune cells of small intestine muscularis
  • Method for quantitatively separating and detecting infiltrative immune cells of small intestine muscularis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Quantitative isolation of small intestinal muscle infiltrating immune cells in an inflammatory state

[0034] 1. Liquid preparation

[0035] a. Lysis medium:

[0036] α-MEM medium (Sigma-Aldrich: M4526): 500ul

[0037] Penicillin-Streptomycin (penicillin-streptomycin double antibody solution, Gibco: 15140122, containing 10000 units / mL of penicillin and 10000 μg / mL of streptomycin): 50ul

[0038] 2-Mercaptoethanol (2-mercaptoethanol, Sigma-Aldrich: M6250): 500ul

[0039] 5% Fetal bovine serum (5% (v / v) fetal bovine serum, Gibco: 10093): 25ml

[0040] b. Digestion Medium

[0041] For every 5ml of lysis medium add:

[0042] 3.13ug DNase I (Sigma-Aldrich: 10104159001)

[0043] 250ug COLLAGENASE II (Collagenase II, Sigma-Aldrich: C5138-100MG)

[0044] 125ug PROTEASE type I (Type I protease, Sigma-Aldrich: 9001-92-7)

[0045] c. Flow buffer

[0046] PBS 500ml

[0047] 2% Fetal bovine serum (Gibco: 10093): 10ml

[0048] 2. Take materials

[0049] Inflamma...

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Abstract

The invention provides a separating and detecting method for the infiltrative immune cells of small intestine muscularis, and belongs to the field of immune cell separating detection. The method disclosed by the invention uses DNase I, collagenase II and I type protease at a specific ratio to carry out enzymolysis to separate cells, and then, a specific antibody combination CD45.2, CD11b, CD64, Ly6G, Ly6C and MHC II is used for carrying out screening to obtain infiltrative mononuclear cells, immature macrophages and mature macrophages. The method disclosed by the invention is the method whichcan separate maximum categories of immune cells from the small intestine muscularis in one time.

Description

technical field [0001] The invention belongs to the field of immune cell separation and detection. Background technique [0002] The small intestine is the longest part of the intestinal tract. It is composed of intestinal villi, intestinal glands, capillaries, lymphatic capillaries, inner circular muscles, and outer longitudinal muscles. It is a multi-layered complex and precise organizational structure. Existing theories believe that the intestine is not only a digestive organ, but also an immune organ, which has a resting regulatory effect on the inflammatory-immune response. Under physiological conditions, immune cells are mainly distributed in the lamina propria of the small intestine. When the small intestine is stimulated by endogenous or exogenous inflammation, immune cells circulating in the periphery will cross the vascular barrier and infiltrate into the muscular layer of the small intestine. [0003] There are very few reports on the isolation of immune cells in...

Claims

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Application Information

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IPC IPC(8): C12N5/078C12N5/0786C12Q1/02
CPCC12N5/0634C12N5/0645G01N33/5005C12N2509/00
Inventor 吴骎
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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