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Improved neutrophil NETs immunofluorescence detection method

An immunofluorescence detection, neutrophil technology, applied in fluorescence/phosphorescence, biological testing, measurement devices, etc., can solve the problem of false positives, and achieve the effect of eliminating false positives and reducing the risk of cells being washed out

Pending Publication Date: 2021-08-27
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] However, detection of neutrophil NETs using immunofluorescence assays is prone to false positives

Method used

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  • Improved neutrophil NETs immunofluorescence detection method
  • Improved neutrophil NETs immunofluorescence detection method
  • Improved neutrophil NETs immunofluorescence detection method

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Experimental program
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Embodiment Construction

[0022] The following describes the substantive content of the present invention in detail in conjunction with the drawings and embodiments, but does not limit the protection scope of the present invention.

[0023] 1. Experimental materials

[0024] Experimental consumables: masks, gloves, caps, disposable venous blood collection needles, 5mL sterile EDTA anticoagulant tubes, adhesive slides, coverslips, 15mL centrifuge tubes, 1.5mLEP tubes, 24-well cell culture plates, immunohistochemical pens , Pasteur pipette, etc.

[0025] Experimental reagents: RPMI-1640 medium, human peripheral blood lymphocyte separation medium, red blood cell lysate, Phorbol12-Myristate 13-Acetate (PMA), 1% paraformaldehyde, Triton-X100, Tween-20, Fluoromount-G fluorescent seal Tablet (containing DAPI nuclear dye), PBS powder, fetal bovine serum albumin, primary antibody (donkey anti-rabbit MPO antibody), secondary antibody (anti-rabbit-594), etc.

[0026] Experimental equipment: SW-CJ-2FD ultra-clea...

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Abstract

The invention discloses an improved neutrophil NETs immunofluorescence detection method. The method is characterized in that normal-temperature drying is performed for 6-12 hours before paraformaldehyde is fixed. In a NETs verification experiment, the improved method can help to eliminate false positive caused by paraformaldehyde immobilization, can obviously reduce the risk that cells are washed off in the experiment process, and is beneficial to development of related experiments of NETs of neutrophils.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an improved neutrophil NETs immunofluorescence detection method. Background technique [0002] Neutrophils are one of the main effector cells of the innate immune system and have various immune functions, such as phagocytosis, degranulation, production of reactive oxygen species, and formation and release of neutrophil extracellular traps (neutrophil extracellulartraps, NETs). NETs is a network structure composed of depolymerized DNA as the skeleton and neutrophil granule protein. The production of NETs is affected by various inducing factors, such as phorbol myristoyl ethyl ester (PMA). [0003] NETs are closely related to the pathogenesis and treatment of many diseases, showing the role of "double-edged sword". On the one hand, it can prevent the spread and kill pathogenic microorganisms in infectious diseases, such as Staphylococcus aureus, Streptococcus pneumoniae, ...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N33/53
CPCG01N21/6428G01N33/53
Inventor 赵毅黎艳红陈桃胡惠方孙蕊
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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