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Method for recognizing mouse intravascular and extravascular lymphocytes and application thereof

A lymphocyte and mouse technology, applied in the field of immunology, can solve the problem of not being able to provide immune cell-related information, and achieve the effect of fewer steps, shorter time, and avoidance of limitations.

Pending Publication Date: 2020-09-25
XUZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, flow cytometry technology can only detect fresh tissue samples and cannot provide relevant information on the distribution of immune cells in the immune microenvironment

Method used

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  • Method for recognizing mouse intravascular and extravascular lymphocytes and application thereof
  • Method for recognizing mouse intravascular and extravascular lymphocytes and application thereof
  • Method for recognizing mouse intravascular and extravascular lymphocytes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 Antibody injection concentration analysis

[0043] 1 material

[0044] 1.1 Experimental animals

[0045] C57BL / 6J mice, 13-16 weeks old, male, SPF grade, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and raised in the IVC system of the Animal Center of Xuzhou Medical University Institute of Blood Diseases (Italy TECNIPLAST company, qualified Certificate number: 13103254).

[0046] 1.2 Main reagents and instruments

[0047] Fluorescence-labeled mouse anti-human monoclonal antibodies CD4-PEcy7 (clone No.RM4-5), CD8-APC (clone No.53-6.7), CD45-BV570 (clone No.30-F11), purchased from Biolegend, USA, CD45-APCcy7 (clone No.30-F11) was purchased from BD Company in the United States, and mouse erythrocyte lysate and staining buffer were prepared by ourselves. The flow cytometry instrument model is FACSCalibur (BD Company, USA), and flowjo v10 software is used for data analysis.

[0048] 2 methods

[0049] 2.1 Intravenous ...

Embodiment 2

[0053] Example 2 Detection of different fluorescently labeled antibodies

[0054] In order to detect whether antibodies from different markers and manufacturers can achieve similar in vivo staining effects, anti mCD45-APCcy7 and anti mCD45-BV570 antibodies with the same clone number (clone No.30-F11) were used for detection. There was no significant difference in the percentage of CD45+ cells in live cells after injecting 3ug / mouse of anti-mCD45-APCcy7 and anti-mCD45-BV570 antibodies respectively. CD4 stained with two different antibodies + T cells and CD8 + 96% of T cells mean CD45 + cells, the non-staining effect was comparable between the two ( figure 2 ). The above results show that even if the CD45 antibodies with the same clone number are labeled with different fluorescence, it does not affect the ability to quickly bind to the antigen in vivo, and the antibody's ability to recognize intravascular lymphocytes is not affected.

Embodiment 3

[0055]Embodiment 3 Lymphocyte labeling situation inside and outside blood vessels in lymphoid organs and non-lymphoid organs

[0056] 1 method

[0057] 1.1 Preparation of mononuclear cells

[0058] Peripheral blood mononuclear cells: prepare EDTA anticoagulant EP tubes, add 50ul 0.5M EDTA to 1.5ml EP tubes, remove the mouse eyeballs and take blood and drop them into EP tubes, shake and bounce immediately to prevent coagulation; micropipette draws 200ul peripheral blood mononuclear cells Transfer the blood to a 15ml centrifuge tube, add 5ml mouse erythrocyte lysate to lyse at room temperature for 6 minutes, add 10ml PBS to the centrifuge tube to stop the lysis, centrifuge at 1100rpm, 8min, 4°C, discard the supernatant, add 1ml PBS to the residual solution and place on ice.

[0059] Preparation of LN and SP mononuclear cells: add PBS to the plate without passing through the tissue in the sieve, grind the lymph node and spleen with the needle core of the syringe until only conne...

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Abstract

The invention discloses a method for recognizing mouse intravascular and extravascular lymphocytes and application thereof. The method comprises the steps of intravenous injection of an anti-CD45 antibody, preparation of mononuclear cells, preparation of a flow sample, analysis of labeling conditions of intravascular and extravascular lymphocytes and the like. Compared with the prior art, the method has the following advantages that (1) multi-label detection of samples can be realized; (2) lymphocyte conditions of a plurality of organs can be simultaneously detected; (3) the experiment resultis reliable; (4) operation steps are simple, and consumed time is short; and (5) the method is very wide in application prospect, and can be used as a factor for judging disease prognosis according tothe severity of lymphocyte infiltration. Lymphocyte infiltration conditions can be analyzed and studied in association with disease or tumor typing, prediction of response of clinical therapy, and regulation and control of a certain link of disease progression.

Description

technical field [0001] The invention belongs to the technical field of immunology, and relates to an efficient, fast and accurate detection method of living mouse lymphocytes, in particular to a method for identifying lymphocytes inside and outside the blood vessels of mice and its application. Background technique [0002] Under physiological conditions, initial T lymphocytes are mainly concentrated in secondary lymphoid organs (Secondary lymphoidorgans, SLOs) such as the spleen and lymph nodes, and travel between blood circulation and lymphatic circulation to play an immune surveillance role, and rarely reside in non-lymphoid organs. Keep. However, when inflammation, tumor or pathological damage occurs in the body, naive T lymphocytes are activated into effector T cells. Under the action of the expressed chemokine receptors, the effector cells infiltrate from blood vessels or lymphatic vessels and distribute in various non-lymphatic organs, including liver, intestinal tra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/58G01N15/14
CPCG01N33/6872G01N33/577G01N33/582G01N33/56972G01N15/14G01N2333/70589
Inventor 赵恺黄栋沈沈徐开林
Owner XUZHOU MEDICAL UNIV
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