Mouse pancreas infiltrated immune cell separation method

A separation method and technology of immune cells, which is applied in the field of separation of immune cells infiltrated in the mouse pancreas, can solve the problems that the immune cells infiltrated in the pancreas cannot be used, the steps are complicated, and the yield is low, and the extraction steps are simplified and the quantity is large. , the effect of high purity

Inactive Publication Date: 2018-01-30
SUZHOU UNIV
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Problems solved by technology

[0003] In view of the shortcomings of the existing separation methods, such as long time-consuming, low yield, complicated steps and inability to be used for infiltrating i

Method used

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  • Mouse pancreas infiltrated immune cell separation method
  • Mouse pancreas infiltrated immune cell separation method
  • Mouse pancreas infiltrated immune cell separation method

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Experimental program
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Embodiment Construction

[0011] 1 Materials and methods

[0012] 1.1 Experimental animals: C57BL / 6 male mice, 6-8 weeks old, purchased from Shanghai Slack Animal Center, and kept in SPF grade animal room.

[0013] 1.2 Main reagents and equipment:

[0014] Reagents: RPMI1640 culture medium (Gibco, USA), fetal bovine serum (ICP, New Zealand), 1XPBS (phosphate buffer saline, 0.01mol / L), anti-mouse CD45 monoclonal antibody, anti-CD3 monoclonal antibody, anti -mouse CD4 monoclonal antibody, anti-mouse CD8 monoclonal antibody, anti-mouse CD19 monoclonal antibody (BD, USA); Collagenase IV digestion solution (50mg / ml); DNase I (1mg / ml);

[0015] Percoll separating fluid, etc.

[0016] Equipment: 15ml, 50ml centrifuge tubes; cell culture dishes; surgical instruments; Eppendorf centrifuge, Niko microscope, BD FACSCantoTM II flow cytometer, etc.

[0017] 1.3 Isolation method of mouse pancreatic lymphocytes

[0018] 1.3.1 Preparation of pancreatic tissue pieces: sacrifice the mice, take out the pancreas, plac...

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Abstract

The invention discloses a mouse pancreas infiltrated immune cell separation method which comprises the following steps: digesting pancreas tissue by using mixed digestive juice of collagenase IV, DNAenzyme I and RPMI1640, filtering by using a nylon filtering net of 100 [mu]m, performing density gradient centrifugation, collecting middle-layer cells after centrifugation, and analyzing cell yield,a survival rate and cell types. Results show that (3.0+/-1.2)*10<5> cells can be collected from each mouse, the survival rate of separated cells is (96.7+/-2.2)%, flow cytometry shows that the contentof CD45 positive T cells is (97.1+/-3.5)%, the content of CD3 positive T cells is (61.2+/-5.7)%, the contents of CD4 positive T cells is (49.1+/-5.1)%, the content of CD8 positive T cells is (47.2+/-5.3)%, and the content of CD19 positive B cells is (32.8+/-4.3)%. The conclusion is that the method is simply, efficient and stable, a great number of cells of a high survival rate and high purity arecollected, and a stable and reliable method is provided for related research on separation of pancreas infiltrated immune cells.

Description

technical field [0001] The present application relates to a method for separating cells in tissues, in particular to a method for separating immune cells infiltrated in mouse pancreas. Background technique [0002] Pancreas is one of the most important organs in the human body, and its physiological functions and pathological changes are closely related to life. In recent years, with the improvement of people's quality of life, people's diet structure has also changed, so more and more pancreatic diseases have appeared. At present, the incidence of pancreatic diseases in my country and even in the world is on the rise. There are more and more reports on pancreatic diseases, such as acute and chronic pancreatitis, idiopathic pancreatitis, autoimmune pancreatitis, and pancreatic cancer, and pancreatic diseases have gradually attracted attention. Many researchers focus on the study of the pancreas on the signal transduction pathways related to pancreatic cancer, the molecular...

Claims

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Application Information

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IPC IPC(8): C12N5/078
Inventor 李敏徐薇颜克鹏卫林
Owner SUZHOU UNIV
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