B Cell activation

Summary

B cells are activated and proliferate upon activation by specific antigens, anti-Ig antibodies, and/or mitogens.

Principle

B The basic principle of cell activation is that activation and proliferation can occur following activation by specific antigens, anti-Ig antibodies, and/or mitogens.

Operation method

B Cell activation

Materials and Instruments

Reagents:
(1) Purified B cells.
(2) Anti-IgM antibody, bacterial LPS (E. coli; Difco #011B4).
(3) Soluble CD40L, anti-mouse or human CD40 antibody.
(4) RPMI 1640 culture medium containing 10% FCS.
Instrumentation:
(1) 48-well flat-ground culture plate.
(2) Flow cytometer

Move

The basic process of B cell activation can be divided into the following steps:

(1) Purified B cells, adjust the B cell concentration to 10/ml with RPMI 1640 culture medium containing 10% FCS. (2) Add 1 ml of B cell suspension to a 48-well flat-bottom plate, i.e. 1x10/well. (3) Add one of the following stimuli to the B cell suspension and set up 3 replicate wells for each condition: (1) Anti-IgM antibody, 2~200μg/ml; 2) LPS, 1~100 μg/ml. 3) sCD40L, 0.03~0.1μg/ml. 4) anti-CD40 antibody, 0.2~0.5μg/ml; (5) negative control without any stimulant. (4) Place the culture plate in a 37℃, 5% _CO2 cell culture incubator for 24-72 hours to obtain activated B cells. (5) Collect the B cells and surface label the activated B cells with FACS method for the expression of surface molecules, such as MHC class II molecules, CD80 and CD86.

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