實驗概要
Isolate B cells from total mouse cells
實驗步驟
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/1X108 cells, and stand in RT for 1 min.
Constant volume to 20 ml/ 1X108 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 1~2X107 cells / ml, Adding LPS(1000X).
13. Pipette cells to 6-well-plate.