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Direct monitoring method of mast cell degranulation

A mast cell, degranulation technique, applied in the field of determination of substances that cause anaphylactoid reactions

Inactive Publication Date: 2011-08-17
EXPERIMENTAL RES CENT CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, directly observing the direction of movement of vesicles and monitoring the process of their fusion with the membrane is undoubtedly the most direct method to dynamically observe degranulation, but there is no suitable method to evaluate it.

Method used

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  • Direct monitoring method of mast cell degranulation
  • Direct monitoring method of mast cell degranulation
  • Direct monitoring method of mast cell degranulation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Embodiment 1, measuring method

[0088]Step 1, the RBL-2H3 cells transfected with CD63-GFP plasmid were cultured with MEM medium (MEM medium: MEM powder 9.4g, L-glutamine 0.292g, NaHCO 3 Add 1000ml of three-distilled water to 2.2g, add 10% fetal bovine serum, 100,000 units of penicillin and streptomycin when used), after three passages, inoculate the cells into a confocal culture dish (diameter 3cm, glass bottom thickness 0.17μm) at 37°C After culturing in the incubator for 24 hours;

[0089] Step 2, replace the culture medium with Tyrode's solution (preparation method: NaCl 400mg, KCl 100mg, MgCl 2 50mg, NaHCO 3 500mg, NaH 2 PO 4 32.5 mg, CaCl 2 100g, add distilled water to 1000ml, add glucose 1.0g before use), incubate at 37°C for 30min;

[0090] Step 3, after adding the test drug, move to a laser scanning confocal microscope for observation under a 100X oil lens. After selecting a field of view with good cell growth and no overlap with each other, images w...

Embodiment 2

[0097] Embodiment 2, allergic substance assay method

[0098] Step 1, the RBL-2H3 cells transfected with CD63-GFP plasmid were cultured with MEM medium (MEM medium: MEM powder 9.4g, L-glutamine 0.292g, NaHCO 3 Add 1000ml of three-distilled water to 2.2g, add 10% fetal bovine serum, 100,000 units of penicillin and streptomycin when used), after three passages, inoculate the cells into a confocal culture dish (diameter 3cm, glass bottom thickness 0.17μm) at 37°C After culturing in the incubator for 24 hours;

[0099] Step 2, replace the culture medium with Tyrode's solution (preparation method: NaCl 400mg, KCl 100mg, MgCl 2 50mg, NaHCO 3 500mg, NaH 2 PO 4 32.5 mg, CaCl 2 100g, add distilled water to 1000ml, add glucose 1.0g before use), incubate at 37°C for 30min;

[0100] Step 3, after adding the suspected allergic substance to be tested, move to a laser scanning confocal microscope for observation under a 100X oil lens. After selecting a field of view with good cel...

Embodiment 3

[0107] Embodiment 3, Chinese medicine injection allergen determination method

[0108] Step 1, the RBL-2H3 cells transfected with CD63-GFP plasmid were cultured with MEM medium (MEM medium: MEM powder 9.4g, L-glutamine 0.292g, NaHCO 3 Add 1000ml of three-distilled water to 2.2g, add 10% fetal bovine serum, 100,000 units of penicillin and streptomycin when used), after three passages, inoculate the cells into a confocal culture dish (diameter 3cm, glass bottom thickness 0.17μm) at 37°C After culturing in the incubator for 24 hours;

[0109] Step 2, replace the culture medium with Tyrode's solution (preparation method: NaCl 400mg, KCl 100mg, MgCl 2 50mg, NaHCO 3 500mg, NaH 2 PO 4 32.5 mg, CaCl 2 100g, add distilled water to 1000ml, add glucose 1.0g before use), incubate at 37°C for 30min;

[0110] Step 3, after adding the traditional Chinese medicine injection, move to a laser scanning confocal microscope for observation under a 100X oil lens. After selecting a field...

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Abstract

The invention belongs to the field of medicine detection, and relates to a method for determining an allergen through directly monitoring mast cell degranulation. The method is characterized by comprising the following steps: step 1: culturing an RBL-2H3 cell transfected with CD63-GFP (green fluorescent protein) plasmid and the allergen together; step 2: scanning and collecting the image of the culture by a microscope; and step 3: determining the existence of the allergen according to the existence and the movement of fluorescent particles displayed in the image.

Description

technical field [0001] The invention belongs to the technical field of medical detection, and in particular relates to a method for measuring substances that cause anaphylactoid reactions. Background technique [0002] Anaphylaxis is one of the common adverse reactions. Anaphylaxis is an abnormal immune reaction between exogenous antigenic substances and antibodies in the body. There are many substances that can induce allergic reactions, such as proteins, polypeptides, polysaccharides and other macromolecular substances with complete antigenicity; other compounds with smaller molecules can be used as haptens to combine with proteins in the body to form complete antigens, thereby causing allergic reactions. At present, there are many allergic substances involved in allergic reactions, among which medicines (traditional Chinese medicine and western medicine), food, pollen, mites, etc. occur more frequently. [0003] The latest research shows that 77% of acute allergic react...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N21/84C12Q1/02
Inventor 王毅王丹巧胡剑江张倩侯燕鸣雷洪涛于友华李连达
Owner EXPERIMENTAL RES CENT CHINA ACAD OF CHINESE MEDICAL SCI
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