B Identification of functional subpopulations of cells

Summary

Peripheral mature B cells can be divided into several subpopulations. Traditionally, B cells have been divided into two subpopulations, CD5+ B1 cells and CD5 B2 cells, according to their phenotype, histological localization, and functional characteristics.The main function of B cells is to secrete antibodies and participate in humoral immunity. Under certain circumstances, B cells can also secrete cytokines and play an immunological regulatory role, and according to the different cytokines secreted, B cells are classified into subpopulations with different effector functions, and regulatory B cells play a role through the production of the negative regulatory factor IL-10.

Principle

The basic principle of the identification of B cell functional subpopulations is to utilize multichannel flow cytometry to detect different B cell subpopulations in the same organ to characterize B cell phenotype and function.

Operation method

B Identification of functional subpopulations of cells

Materials and Instruments

Reagents:
(1) Brefeldin A (BFA) or monensin.
(2) Fc blocker.
(3) 1x phosphate buffer (1xPBS).
(4) Staining buffer (1xPBS + 0.1% BSA + 0.05% sodium azide).
(5) Cell fixation solution (4% paraformaldehyde).
(6) membrane-breaking solution (1xPBS + 0.1% bovine serum albumin + 0.05% sodium azide + 0.1% saponin).
(7) RPMI 1640 culture medium containing 10% FCS, PBS containing 5% FCS.
(8) Fluorescently labeled mAb.
Instrumentation:
Flow cytometer.

Move

The basic process of B cell functional subpopulation identification can be divided into the following steps:
(1) Preparation of cell suspension: resuspend the cells with RPMI 1640 culture medium containing 10% FCS, adjust the concentration of the cells to ^2x106/ml, and then culture them in vitro.
(2) In vitro cell culture
1) For the selection of specific stimulants, refer to B cell activation conditions, or stimulate the cells with specific antigens.
2) Set up a negative control group without any stimulants.
3) Add the blocking agent BFA to each culture condition (or monensin for the detection of IL-10).
4) Incubate the cells in an incubator for 6 hrs. at 37℃, 5% _{CO2. incubator} for more than 6 hours.
(3) Fluorescein antibody labeling of cells:
1) Surface molecular labeling: resuspend 0. ^5x106--1x106 cells in 100μl of staining buffer, add Fc blocker (CD16/CD32) before labeling to prevent Fc receptors from binding to the antibody; according to the experimental design scheme, add a sufficient amount of According to the experimental design, add sufficient amount of fluorescein-labeled antibodies against surface molecules to the cell suspension; incubate at 4℃ for 30 minutes, avoiding light; wash twice with staining buffer.
2) Fixation of cells: Discard the supernatant, add 4% paraformaldehyde, 500 μl/tube, mix well, and avoid light for 8 minutes at room temperature; wash twice with staining buffer.
3) Breaking of membranes: Discard the supernatant, and resuspend the cells with membrane-breaking solution (no need to fix and break the membranes if only the surface molecules of the cells are analyzed). The cells should be washed with staining buffer for 2 times.
4) Resuspend the cells with staining buffer, 100 μl/tube; according to the experimental design, add the corresponding staining antibody, mix well, and incubate for 30 minutes at 4℃ away from light; wash with staining buffer for 2 times; discard the supernatant, and wash with 150--200 μl of wash buffer; discard the supernatant, and wash with 150--200 μl of wash buffer. -200 μl of wash solution resuspended, placed at 4℃ away from light, waiting for the machine.
5) On the flow cytometer to obtain data and analyze the data.

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