Detection method for adhesion rate and phagocytosis rate of phagocytes on baterial

A technology of phagocytic cells and detection methods, applied in measurement devices, particle and sedimentation analysis, individual particle analysis, etc., can solve the problems of manpower, material resources, affecting scientific research efficiency, increasing scientific research burden, etc., achieving less reagents and reducing scientific research expenditures , the effect of price economy

Inactive Publication Date: 2017-02-22
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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  • Abstract
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  • Application Information

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Problems solved by technology

Compared with the present invention, the existing method has obvious deficiencies. Firstly, the above-mentioned method takes a long time and consumes manpower and material resources; secondly, it is not economical to purchase more expensive reagents such as primary antibody and secondary antibody; and it needs multiple preparations. Experimentally determine the optimal dilution of antibodies, increasing the burden of scientific research affects scientific research efficiency

Method used

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  • Detection method for adhesion rate and phagocytosis rate of phagocytes on baterial
  • Detection method for adhesion rate and phagocytosis rate of phagocytes on baterial
  • Detection method for adhesion rate and phagocytosis rate of phagocytes on baterial

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The study found that incubation of 0.4% trypan blue with CFDA-SE-labeled bacteria at room temperature for 5 min could effectively quench the fluorescence emitted by CFSE ( figure 1 ).

[0025] 1) Experimental grouping

[0026] The experiment was divided into three groups: control group, 0.4% trypan blue treatment group and 0.4% trypan blue untreated group. Because the cells have autofluorescence and the fluorescence value of CFSE quenched by trypan blue is higher than that of unlabeled cells ( figure 1 C), so the cells in the control group were pretreated with cytochalasin D (20ug / ml) for 30 minutes to inhibit the phagocytosis, but it did not inhibit the adhesion of bacteria to the cells.

[0027] 2) CFDA-SE labeled bacteria

[0028] Bacteria cultured to the logarithmic phase were washed with phosphate buffered saline (PBS) and the concentration was adjusted to 10 8 / mL, add CFDA-SE (Sigma-Aldrich, USA) to make the working concentration 5μM, incubate at room tempera...

Embodiment 2

[0034] 1) Experimental grouping

[0035] The experiment was divided into three groups: control group, cytochalasin D treatment group and cytochalasin D untreated group. Control group is treated with embodiment 1. Cells in cytochalasin D treatment group were pretreated with cytochalasin D (20ug / ml) for 30min to inhibit the phagocytosis.

[0036] 2) CFDA-SE labeled bacteria

[0037] With embodiment 1.

[0038] 3) Interaction between bacteria and cells

[0039] The prepared bacteria were co-incubated with each group of phagocytes at an appropriate MOI (proportion of bacterial cells). After the incubation was completed, the treatment of the control group was the same as in Example 1, adding 0.4% trypan blue and incubating at room temperature for 5 minutes.

[0040] 4) Flow cytometry detection

[0041] The control group was used as the gate standard for flow cytometry detection. The test result of the cytochalasin D untreated group was the sum of the adhesion rate and the phago...

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Abstract

The invention discloses a detection method for the adhesion rate and the phagocytosis rate of phagocytes on bacterial. According to the detection method, phagocytes are divided into a control group, a treatment group and a untreated group, the control group cells are treated with trypan blue and cytochalasin D, the treatment group cells are treated with trypan blue or cytochalasin D, the untreated group cells are not treated, bacteria are labeled with CFDA-SE, and after the bacterial and cells interact, the adhesion rate and the phagocytosis rate of the phagocytes are detected through flow cytometry. According to the present invention, the adhesion rate and the phagocytosis rate of the phagocytes on pathogenic bacteria can be rapidly and accurately detected, and the whole experimental process does not exceed 3 h; the required operation steps after the interaction of the pathogenic bacteria and the phagocytes are less, such that the detection of the large batch sample can be performed; the required reagent is less and the price is economical so as to substantially reduce the scientific research cost; and the detection method is suitable for the adherently growing phagocytes and the difficult-detected phagocytes having the suspending growing state, and broaden the research idea to a certain extent.

Description

technical field [0001] The invention belongs to the field of biology, and more specifically relates to a method for detecting the adhesion rate and phagocytosis rate of phagocytic cells to bacteria. Background technique [0002] In the body's immune system, innate immunity plays a very important role, especially in the early stage of infection, which plays a decisive role in controlling the spread of infection. In innate immunity, monocyte-macrophages are the most effective phagocytes, which are responsible for the important function of the body's resistance to foreign pathogens. Studying the interaction between pathogenic bacteria and phagocytes is very important for elucidating the pathogenesis of infectious diseases. Determining the adhesion rate and phagocytosis rate of phagocytes to pathogenic bacteria is the first step in the study of the interaction between the two, which is of great significance for exploring the adhesion receptors, phagocytosis receptors and immune...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/14
Inventor 张彦何平郭晓奎刘伯玉王砚春
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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