B cells play a central role in immune development. They initially enter the bloodstream from the bone marrow as naive B cells, which can migrate to lymphoid tissues such as the spleen, lymph nodes, and tonsils for further development. Some naive B cells migrate to lymphoid follicles, where germinal center B cells can differentiate into memory B cells and plasmablasts (PBs)/plasma cells (PCs). Although most PBs/PCs eventually enter the bloodstream, a small number ultimately reside in the bone marrow and undergo terminal differentiation into long-lived plasma cells.

Let's start with the extraction of B cells:

• Collect 2 ml of venous blood into a test tube containing heparin solution (50 μg/ml blood sample), mix well to anticoagulate the blood, and dilute with PBS at a ratio of 1:1.

• Pipette 2 ml of lymphocyte separation medium (Qida Biotech, Cat. No.: SD0059) into a graduated centrifuge tube. Tilt the tube at 45°, and slowly add the diluted whole blood onto the separation medium along the tube wall using a capillary pipette, taking care to maintain a clear interface between the two layers.

• Centrifuge at 1500 r/min for 20 min at 18–20 ℃ using a horizontal centrifuge.

• Carefully remove the centrifuge tube and slowly aspirate the lymphocytes in the second layer with a 1 ml pipette. Balance the tubes and centrifuge again. If red blood cells remain, wash an additional time with PBS.

• Discard the supernatant, and resuspend the pelleted cells in culture medium for subsequent use.

B Cell Purification

(a) Transfer 1 mg of carboxyl magnetic beads (approx. 100 μl bead suspension; mix thoroughly by ultrasonication before use) into a 1.5 ml Eppendorf tube, and vortex for 5–10 min.

(b) Take an appropriate volume of bead suspension, resuspend the cells in 20 volumes of PBS + 0.2% BSA solution, then place on a magnetic stand for 5 min until the beads are fully adsorbed.

Note: The ratio of magnetic beads to mononuclear cells or whole blood is 0.5 mg beads per 1×10⁷ mononuclear cells or per 1 ml whole blood.

(c) Carefully aspirate the supernatant with a pipette to remove the secondary antibody detached from the beads during storage. Resuspend the beads in 100 μl of PBS + 0.2% BSA solution. Add the primary antibody at a ratio of 5 μg primary antibody per 1 mg magnetic beads, mix well, and incubate for 30 min at room temperature on a sample mixer for activation.

(d) Add 2 ml of PBS + 0.2% BSA solution, place on a magnetic stand for 5 min, then aspirate the supernatant to remove unbound primary antibody. Repeat this step once. Resuspend the beads in 100 μl of PBS + 0.2% BSA solution.

(e) Adjust the concentration of cells to be separated to 1×10⁷ cells/ml using complete B cell medium (Qida Biotech, Cat. No.: P3901). Add the cell suspension to the bead mixture, incubate at room temperature for 15 min with gentle mixing once halfway, then place on a magnetic stand for 10 min. Aspirate the supernatant.

(f) Resuspend the bead-bound cells in complete B cell medium and use directly for culture.

Alright, check the figure below — B cell isolation was successful!

                                           Day 1 of B cell culture     Day 7 of B cell culture