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Fluorescence kit for detecting cytophagy and preparation method of fluorescence kit

A fluorescence kit and phagocytosis technology, applied in the fields of molecular biology and medicine, can solve the problems of inability to obtain phagocyte morphological images, unfavorable basic laboratory scientific research, and poor suspension cell detection results, and achieve comprehensive and accurate results. Reliable, efficient utilization, long-term preservation effect

Inactive Publication Date: 2018-07-20
王洵
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Existing traditional methods for detecting cell phagocytosis include phagocytosis of neutral red method, phagocytosis of chicken red blood cell method, etc., but there are problems such as limited sensitivity and difficult preservation of reagents. There are kits for detecting cell phagocytosis by fluorescence method abroad, although the sensitivity is limited. However, there are certain defects, such as a narrow application range, it can only be used to measure the phagocytosis efficiency of adherent cells, and the detection effect on suspension cells such as neutrophils is not good; the detection method is single, and only a fluorescent microplate reader can be used However, the morphological images of phagocytic cells cannot be obtained for detection; the fluorescent particles in the kit are only one type of fluorescently labeled E. scientific research

Method used

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  • Fluorescence kit for detecting cytophagy and preparation method of fluorescence kit
  • Fluorescence kit for detecting cytophagy and preparation method of fluorescence kit
  • Fluorescence kit for detecting cytophagy and preparation method of fluorescence kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: the preparation of fluorescence kit

[0047]1. Materials:

[0048] Escherichia coli (ATCC standard strain 25922), Pseudomonas aeruginosa (ATCC standard strain 27853), sodium bicarbonate, sodium hydroxide, and sodium chloride were purchased from Shanghai Sinopharm Group Chemical Co., Ltd., all of which were analytically pure, FITC (isosulfur Fluorescein cyanate) was purchased from SIGMA Company, BSA, 10× concentrated Hanks balanced salt buffer (the specific components were NaCl 137.93mM; KCl 5.33mM; NaHCO3 4.17mM; KH2PO4 0.441mM; Na2HPO4 0.338mM; D-Glucose 5.56mM) , HEPES buffer, 10 mg / ml EB (ethidium bromide) stock solution, trypan blue, and citric acid buffer solution were all purchased from Shanghai Sangon Bioengineering Co., Ltd.

[0049] 2. Reagent preparation:

[0050] FITC solution: every 100ml of basic solution includes 4.2g of sodium bicarbonate, 2g of sodium hydroxide, and 5.84g of sodium chloride, dilute to 100ml with water, the final concentra...

Embodiment 2

[0074] Example 2: 96-well plate fluorescent microplate reader to detect phagocytosis effect experiment

[0075] 1. Self-prepared items:

[0076] Cell line (primary cultured mouse peritoneal macrophages, see Figure 6 ) and penicillin, DMEM medium, hemocytometer, deionized water, cell culture incubator, ultrasonic breaker, 96-well plate, fluorescent microplate reader, flow cytometer, fluorescent microscope, glass slide, cover glass slides, glass bottom Petri dishes, confocal microscope, sterile PBS buffer, trypsin containing EDTA.

[0077] 2. Adopt the fluorescent kit prepared in Example 1 (comprising a fluorescently labeled Escherichia coli lyophilized powder, a Pseudomonas aeruginosa lyophilized powder, a trypan blue solution for quenching excess fluorescence, and a lyophilized powder for dissolving Bacterial Hanks balanced salt buffer and EB solution for nuclear staining), according to the following experimental steps:

[0078] 1. Preparation of cells and fluorescently la...

Embodiment 3

[0097] Example 3: Experiment of phagocytosis effect detected by flow cytometry

[0098] 1. Self-prepared items: the same as in Example 2;

[0099] Two, adopt the fluorescence kit prepared in embodiment 1, carry out according to the following experimental steps:

[0100] 1. Preparation of cells and fluorescently labeled cells: the steps are the same as in Example 2;

[0101] 2. Determination of cell viability: the steps are the same as in Example 2;

[0102] 3. Prepare fluorescently labeled bacterial suspension: the steps are the same as in Example 2;

[0103] 4. Prepare the experimental and control groups:

[0104] 4.1 Set up a positive control group and an experimental group, in order to reduce errors, at least 5 duplicate holes for each sample;

[0105] 4.2 Positive control group: add 300ul cell suspension + 150ul DMEM to each microwell;

[0106] 4.3 Experimental group: add 300ul cell suspension + 150ul experimental drug solution to stimulate phagocytosis in each microw...

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Abstract

The invention relates to a fluorescence kit for detecting cytophagy and a preparation method of the fluorescence kit, and belongs to the fields of molecular biology and medicine. The fluorescence kitconsists of fluorescence labeled escherichia coli, and the fluorescence kit is characterized by further consisting of fluorescence labeled pseudomonas aeruginosa. The preparation method of the fluorescent kit comprises the following steps: 1, implementing amplification and concentration detection of escherichia coli / pseudomonas aeruginosa; 2, implementing fluorescence labeling on the escherichia coli; and 3, preparing the fluorescence labeled pseudomonas aeruginosa. The fluorescence kit provided by the invention, which consists of the fluorescence labeled escherichia coli and the fluorescencelabeled pseudomonas aeruginosa which can be engulfed by cells, can judge bacterium number of the cytophagy by detecting the strength of fluorescence signals in the cells, so that phagocytic activity of the cells can be determined.

Description

technical field [0001] The invention relates to a fluorescent reagent kit for detecting cell phagocytosis and a preparation method thereof, belonging to the fields of molecular biology and medicine. Background technique [0002] Phagocytosis is the oldest and one of the most basic defense mechanisms of organisms. Phagocytes have the function of phagocytosis and digestion of foreign bodies. When pathogenic microorganisms invade the body, they can be phagocytized and eliminated by phagocytes before the immune response is stimulated, which is of great significance in the body's non-specific immunity. Different gene expressions, cytokines, drugs, etc. can change the phagocytic function of cells and affect the body's non-specific immunity. Therefore, measuring the phagocytic ability of phagocytic cells has very important scientific research and clinical significance. [0003] Existing traditional methods for detecting cell phagocytosis include phagocytosis of neutral red method,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02C12R1/19C12R1/385
CPCG01N21/6428G01N2021/6432
Inventor 王洵强鑫垚刘怿君
Owner 王洵
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