Separation of T cells and B cells by magnetic bead method
Separation of T cells and B cells by magnetic bead method
This unit describes the separation of T cells, B cells, and T cell subpopulations by magnetic separation techniques. Author: J.E. Collier et al, Translator: Xuitao Cao et al, This experiment is from "Comprehensive Immunology Laboratory Guide".
Operation method
Separation of T-cells and B-cells by magnetic bead method Move Option 1 Automated separation of total T cells, CD4+ cells or CD8+ T cells using the AUTOMACS system. Materials mice HBSS Wash Buffer mice HBSS Wash Buffer MACS Buffer CD45R (B2 2 0 ) beads (Miltenyi) RPMI-10 complete medium autoMACS system and sorting columns (Miltenyi) 30um nylon mesh (BD) or 40/xm pre-separation membrane (Miltenyi) 1 . Prepare a single-cell suspension from the spleens of 2 mice and remove the erythrocytes (Unit 2.1). 2. Cells are suspended in 10 ml of HBSS wash buffer and counted using a blood cell counter (Appendix 3A). 3. The cell suspension was centrifuged at 4°C for 10 min at 20 oz. The supernatant was discarded and the precipitate was suspended at a ratio of 0.9 ml MACS buffer per 108 cells. Add 0.1ml CD45R(B220) beads per 108 cells and incubate at 4℃ for 15 min. 4. Follow steps 4 to 7 in Basic Protocol 1 so that B cells will be eluted from the positive channel. MATERIALS HBSS Wash Buffer MACS Buffer CD90 (Thyl.2 ) magnetic beads (Miltenyi) RPMI-IO complete medium autoMACS system and sorting columns (Miltenyi) 30um nylon mesh (BD) or 40um pre-separated membrane (Miltenyi) 1. Prepare a single-cell suspension from the spleens of 2 mice and remove the erythrocytes (Module 2.1). 2. Cells were suspended in IOml HBSS wash buffer and counted using a blood cell counter (Appendix 3A). 3. The cell suspension was centrifuged at 4°C for lOmin at 200 g. The supernatant was discarded and the precipitate was suspended at a rate of 0.9 ml MACS buffer per IO8 cells. Add 0.1 ml of CD90 (Thy1.2) beads per 108 cells and incubate at 4°C for 15 min. 4. Except for the depletes program (not the possel program), follow steps 4~7 in the basic protocol 1 so that helper cells will be eluted from the negative channel. Additional materials (see Basic Option 1 for additional materials) Gas-free MACS buffer, 4°C (keep cold during loading and column wash) Midi MACS Separation Magnet, MACS Multi-Rack, LS or LD Sorting (Miltenyi) 15 ml tubes, sterile la. For isolation of total T-cells, CD4+ T-cells or CD8+ T-cells: prepare and label single-cell suspensions as described above (see Basic Protocol 1, steps 1 to 3). lb. For isolation of B cells: Prepare and label single cell suspensions as described above (see Basic Plan 2, steps 1 to 3). lc. For isolation of helper cells: prepare and label a single cell suspension as described above (see Basic Plan 3, steps 1 to 3). 2. Centrifuge the cell suspension at 4°C, 200 g for lOmin. 3. During centrifugation, attach the Midi MACS Separation Magnet to the MACS Multi Rack and place the LS Sorting Column (for separation of total T-cells, CD4+ T-cells, CD8+ T-cells, or B-cells) or the LD Column (for separation of helper cells) into the magnet, placing a sterile 15 ml centrifuge tube under the sorting column. Rinse the column 3 times with 3 ml of gas-free MACS buffer at 4°C. Discard the eluate and place a clean, sterile centrifuge tube under the column. The negative cells ( CD4+CD2 5- ) obtained by this method can be used as reaction or control cells. Additional materials (see basic option 4 for additional materials) Midi MACS Separation Magnet (Miltenyi) MACS Multi-Use Stand (Miltenyi) LD Sorting Column (Miltenyi) For more product details, please visit Aladdin Scientific website.
mice
Gas-free MACS buffer, 4°C (keep cold during loading and column wash)
