There are many membrane molecules on the surface of B cells, including cell surface differentiation antigen and model immunoglobulin (smlg). At different stages of differentiation and at different settlement locations, B cells express different surface molecules, and thus the stage of differentiation and functional status of B cells can be determined based on the differences in surface molecules.

Principle

The basic principle of B cell phenotype detection is that there are many membrane molecules on the surface of B cells, including cell surface differentiation antigen and model immunoglobulin (smlg). At different stages of differentiation and different settlement locations, B cells express different surface molecules, and thus the stage of B cell differentiation and functional status can be determined based on the differences in surface molecules.

Operation method

Multicolor flow cytometry to detect B-cell phenotypes

Principle

The basic principle of B cell phenotyping assay is to use B cell surface membrane molecules, including cell surface differentiation antigen and model immunoglobulin (smlg). At different stages of differentiation and different settlement locations, B cells express different surface molecules, and thus the stage of differentiation and functional status of B cells can be determined based on the differences in surface molecules.

Materials and Instruments

Reagents:
(1) NP-KLH
(2) Ribi adjuvant
(3) 0.14 mol/L NH, Cl, Hanks solution
(4) Staining buffer (1xPBS + 0.1% BSA + 0.05% sodium azide)
(5) Brefeldin A (BFA) or Monensin
(6) mAb
Instruments:
(1) Flow cytometer
(2) Centrifuge
(3) Filters, syringes, etc.

Move

The basic process of B cell phenotypic detection can be divided into the following steps (using mice as an example, mainly elaborating on the application of multicolor flow cytometry to detect surface molecules of B cells, analyze the phenotype and function of B cells, and identify the different stages of B cell differentiation as well as subpopulations of memory B cells). :
(1) Prepare the single nucleated cell suspension required for the detection of B cells
1

) Immunize the mice:

① Immunize 6-10 week old C57BL/6J mice intraperitoneally with 400 μg NP-KLH dissolved in Ribi adjuvant;

(ii) At least 6 weeks after the initial injection, immunize the mice again by the same method;

(iii) The mice can be executed after 1 week of booster immunization to detect B cells.

) Prepare the cell suspension:

Execute the mice by cervical dislocation method, take the spleen, lysed erythrocytes after filter grinding, resuspended with Hanks liquid, and adjusted the cell concentration to 2x106/ml.

Select appropriate stimulants to stimulate cells in vitro, specifically activate B cells; if you want to detect the expression of cytokines, add the blocking agent BFA at the same time (when detecting IL-10, use monensin).

(3) Fluorescent antibody labeling of cells

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