B Phenotypic detection of cells

Summary

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.

2

) 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.

(2) Culturing cells in vit ro

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

(1) Wash the cells with staining buffer, resuspend the cells at a concentration of 2x10/ml, add the appropriate concentration of biotin labeling mAb, and incubate at 4 ℃ for 30 minutes.
(2) After incubation, wash the cells with staining buffer for 2 times, resuspend the cells in labeling solution containing the appropriate concentration of streptavidin-Cy7APC, and incubate for 15 minutes at 4 ℃ in the refrigerator.
(3) After incubation, wash the cells with staining buffer for 2 times, resuspend the cells in 100 μl of staining buffer containing 2 μg/ml PI (used to exclude the dead cells), and then incubate in an ice bath for no more than 15 minutes to prepare for testing.
(4) Acquire the data on the flow cytometer and analyze the data.

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