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Method for identifying function and capability of cells for phagocytizing exogenous foreign bodies

A technology of cells and foreign bodies, applied in the fields of biochemical equipment and methods, determination/inspection of microorganisms, fluorescence/phosphorescence, etc.

Inactive Publication Date: 2011-11-23
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a method for identifying whether cells have the function of phagocytizing foreign bodies and studying the ability of cells to phagocytize foreign bodies in view of the problems existing in the infection sources used in the existing research on phagocytosis

Method used

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  • Method for identifying function and capability of cells for phagocytizing exogenous foreign bodies
  • Method for identifying function and capability of cells for phagocytizing exogenous foreign bodies
  • Method for identifying function and capability of cells for phagocytizing exogenous foreign bodies

Examples

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Embodiment 1

[0027] Phagocytosis of Escherichia coli expressing green fluorescent protein by Procambarus clarkii hemolymphocytes cultured in vitro.

[0028] 1. Construct Escherichia coli expressing green fluorescent protein:

[0029] a. Extract and purify pEGFP from E.coli DH5α transformed with plasmid pEGFP with B-type mini-plasmid rapid extraction kit (Broadtech), and store at -20°C;

[0030] b. Prepare competent cells (CaCl 2 Law).

[0031] c. Add 200 μl competent cells to 1 μl EGFP plasmid, shake gently, place on ice for 30 minutes, heat shock at 42°C for 90 seconds, place on ice for 2 minutes, add 800 μl LB medium, shake at 200 rpm at 37°C for 1 hour, and centrifuge at 4000 rpm at room temperature After 5 minutes, discard most of the supernatant and resuspend the cells with the remaining 50 μl medium. Bacteria were spread on plates containing 100 µl of X-gal coated ampicillin (100 µg / ml) at a concentration of 20 mg / ml and grown overnight. →Pick positive monoclonal colonies and cul...

Embodiment 2

[0036] Escherichia coli infection in Ciona in vitro

[0037] Cultivate Ciona blood lymphocytes in vitro, (medium formulation 80% Leibovitz L-15, FBS15%, add glucose with a final concentration of 1g / l, 300U of penicillin, streptomycin, 0.1mg / l Vc, 20mMHepes, 0.75g / l KCl, 1.1g / l CaCl2, 2g / l MgCl2, NaCl20g / l, 7.5%NaCO3 adjust the pH value,), all the other are the same as embodiment 1, see specifically figure 2 .

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Abstract

The invention discloses a method for identifying and researching the function and capability of cells for phagocytizing exogenous foreign bodies. The method comprises the following steps: preparing an infection source; performing mixed culture; directly observing the whole process that the researched cells phagocytize the infection source and cysts under a fluorescence microscope; and analyzing changes of phagocytic capability of the researched cells under different conditions so as to obtain phagocytic indexes. The method is characterized in that in the step of preparing the infection source, escherichia coli for expressing green fluorescent protein is taken as the infection source. In the method, the escherichia coli capable of expressing the green fluorescent protein is taken as the infection source so as to research the phagocytosis of various cells; and the method is intuitive, accurate, fast and convenient and can be also used for distinguishing the types of cells with a phagocytic function.

Description

technical field [0001] The invention relates to a method for identifying whether cells have the function of phagocytizing foreign bodies and studying the ability of cells to phagocytize foreign bodies. Background technique [0002] Phagocytosis refers to the process by which cells transport invading pathogens (such as bacteria and viruses) into cells and eliminate them. This process constitutes the first line of defense of organisms' innate immunity. [0003] Phagocytosis is a primitive defense mechanism of all metazoans. As early as 1862, Haeckel described the process of phagocytosis of gastropod blood cells, but the importance of phagocytosis in immune defense was not realized until 1882 by Metchnikoff (Vaughan, 1965). Phagocytosis is ubiquitous in the animal kingdom. Low-level single-celled animals take food through phagocytosis, while higher multicellular animals use phagocytes to remove foreign substances. The phagocytosis process can be summarized as: recognition, adh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/06C12Q1/02G01N21/64
Inventor 刘利平乐亚玲陈桃英
Owner SHANGHAI OCEAN UNIV
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