Mouse B cell isolation with magnetic beads

实验概要 Isolate B cells from total mouse cells   Praparation: 1.Enough MACS; put RPMI into water bath. 2.Put some MACS in 15 ml tube or 1.5 ml tube from cell room, and regulate the centrifuge to 4℃. Step: 1.Get tissue/organ from mice; 2.Place tissue on the cell strainer put on the dish with enough MACS buffer (cover the bottom of the strainer); 3. Grind :Using a syringe plunger, grind the tissue until no big tissue chunks are left and most cells have gone into suspension; 4.Rinse the strainer with MACS buffer, and pipette up and down the cell suspension to break cell aggregates; 5.Transfer cell suspension to 15ml conical tube; 6.Centrifuge cells at 1700 rpm(500 g), 5 min, 4°C; 7. Remove red blood cell : Resuspend cells in ACK to 1 ml/1X10 8 cells, and stand in RT for 1 min. Constant volume to 20 ml/ 1X10 8 cells with MACS, and transfer to another tube through a new strainer, centrifuging for 5 min, 4℃; 8. Isolation : l Positive ：1.Resuspend cells with MACS to 450 ul each spleen; 2.Add 50ul CD19 Microbeads each spleen and incubate on ice for 15min; l Negative : 1.Resuspend cells with MACS to 400 ul and 80 ul B cell Biotin each spleen,incubate on ice for 15 min; 2. Add 300 MACS and 200 anti-biotin Beads each spleen, incubate on ice for 15 min. 9.Constant volume to 20 ml/ each spleen with MACS, and transfer to another tube through a new strainer, centrifuging for 5 min, 4℃; Resuspend cells with MACS to 1 ml/ each spleen. 10.Follow Miltenyi Biotec protocol of column separation Positive :3 ml MACS for equilibrium, then cell solution, then wash 3x3ml followed by 2x4ml . Put the column on a 15 ml tube and flush the column with 5ml; Negative : 3 ml MACS for equilibrium, then collect the elution with 15 ml tube, then cell solution, then wash 3x3ml. 11.Count cells, centrifuging cells at 1700 rpm, 5 min, 4°C. 12.Resuspend with RPMI to 1~2X10 7 cells / ml, Adding LPS(1000X). 13.Pipette cells to 6-well-plate.