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Phagocyte function detection method based on flow cytometry

A technology of flow cytometry and phagocytes, which is applied in the field of flow cytometry-based evaluation of the phagocytosis of phagocytes, can solve the problems of unfavorable promotion and use, weak fluorescence of FITC, and difficulty in distinguishing detection, etc. Simple and easy cost, avoid the effect of cumbersome operation

Active Publication Date: 2020-11-03
SHANDONG UNIV
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Problems solved by technology

At present, there are not many reports on the detection of phagocytic function of phagocytic cells. It has been reported to use fluorescein FITC-labeled bacteria prepared in carbonate buffer to detect phagocytic function of phagocytic cells. However, this method has the disadvantages of weak fluorescence of FITC and difficult to distinguish detection. Not conducive to promotion

Method used

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  • Phagocyte function detection method based on flow cytometry
  • Phagocyte function detection method based on flow cytometry

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Embodiment 1

[0037] 1. Take BALB / c mouse peritoneal macrophages, count them, take 1 million cells, inoculate them in a cell culture plate for culture; take BALB / c mouse bone marrow cells, use Percoll density gradient centrifugation to separate neutrophils, Count and take a certain number of cells.

[0038] 2. Add 100 μL of thiazole orange staining solution to the E. coli bacteria solution, the concentration of the thiazole orange staining solution is 0.5 mg / mL, incubate with shaking at 37°C for 60 minutes, wash, centrifuge, and discard the supernatant;

[0039] 3. Add 5ml PBS to the bottom bacterial pellet after centrifugation, and count and analyze by flow cytometer;

[0040] 4. Add 200 μL of E. coli bacteria stained with Thiazole Orange to the phagocyte culture medium, and incubate for 20 minutes;

[0041] 5. Digest and collect phagocytic cells, add 0.1ml PBS, flow cytometry analysis, thiazole orange excitation light 488nm, emission light 530nm;

[0042] 6. Collect 10,000 cells, analyze the cell ...

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Abstract

The invention discloses a phagocyte function detection method based on flow cytometry, which comprises the following steps of: oscillating and incubating escherichia coli liquid and thiazole orange staining liquid in a constant-temperature shaking table for a proper time, washing off non-specific adhesion by using PBS (Phosphate Buffer Solution), performing centrifuging, and discarding supernate;and finally, adding a proper amount of PBS (phosphate buffer solution) for resuspension, adding into a phagocyte culture solution, and performing culturing in an incubator for a proper time; and digesting and collecting phagocytes, and quantitatively analyzing the cell distribution of the fluorescent population on a flow cytometer, thereby analyzing the phagocytic bacteria capturing function of the phagocytes. The method is rapid, accurate, simple, easy to implement, low in cost and beneficial to application and popularization.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for evaluating the ability of phagocytic cells to engulf bacteria based on flow cytometry. Background technique [0002] Disclosure of the background information is only intended to increase the understanding of the overall background of the present invention, and is not necessarily regarded as an acknowledgement or in any form suggesting that the information constitutes the prior art known to those of ordinary skill in the art. [0003] Phagocytes are a group of cells with phagocytic function in the body, mainly including the mononuclear phagocyte system and neutrophils. The mononuclear phagocyte system includes monocytes that are free in the blood and macrophages that develop after entering various tissues. Among them, macrophages have strong phagocytic ability and are also a major class of antigen presenting cells, which play a key role in the induction and regulatio...

Claims

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Application Information

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IPC IPC(8): G01N15/14G01N21/64
CPCG01N15/1434G01N21/6402
Inventor 赵云雪荆卫强王甘雨毕玉璇韩丽辉安杰郭兴
Owner SHANDONG UNIV
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