B cell isolation

1. Place the spleen in a 6 cm Petri dish containing 1 mL of RPMI-1640 containing 5% FBS.

2. Smash spleen thoroughly between frosted glass slides by placing the spleen on the rough side of the frosted glass slide.

3. Add 1mL of RPMI-1640 containing 5% FBS into the dish, and then taransfer cell suspention from the dish into 5mL tube.

4. Wash the dish by 1mL of RPMI-1640 containing 5% FBS.

5. Spin down the tube at 1400 rpm for 5min at 4℃.

6. Resuspend the cell pelet in 100uL of Biotin cocktail.

・Biotin cocktail

Biotin-conjugated Ab	CD4	CD8	CD11b	CD43	DX5	Ter119
Volume (uL)	0.2	0.2	0.5	0.6	0.2	0.5	/100uL of MACS buffer

MACS buffer: PBS containing 0.5% BSA and 2mM EDTA

7. Incubate cells for 20 min at 4℃.

8. Wash twice by MACS buffer.

Wash: Add 10mL of MACS buffer, spin down the tube at 1400 rpm for 5min at 4℃, and then aspirate supernatant.

9. Resuspend the cell pelet in 100uL of streptavidin beads.

10. Incubate cells for 20 min at 4℃.

11. Negative selection by IMag, and then negative selection by MACS.

IMag separation should be repeated twice.

Naïve T cell isolation

Same as above up to 5. (but transfer cell suspention into 15mL tube).

6. Resuspend the cell pelet in 1mL of ACK lysis buffer to lyse red blood cells.

7. Incubate cells for 1 min 50 sec at room temperature.

8. Add cold MACS buffer up to 10mL.

9. Spin down the tube at 1400 rpm for 5min at 4℃.

10. Resuspend the cell pelet in 500uL of Antibody mix solution.

・Antibody mix solution

11. Incubate cells for 20 min at 4℃.

12. Wash twice by MACS buffer.

13. Resuspend the cell pelet in MACS buffer, and then add 10uL of 10ug/mL Propidium Iodide.

14. Sort CD4+ CD25- CD62L+ CD44- cells by FACSAria.

Equipments

Product name	Manufacturer	Cat #
BD IMagnet Cell Separation Magnet (5mL tube×6 or 14mL tube×2)	BD	552311
MidiMACS™ Separator	Milteny	130-042-302
LS Columns	Milteny	130-042-401
BD FACSAria III cell sorter	BD

Materials

Product name	Manufacturer	Cat #
Biotin anti-CD4	Biolegend	100404
Biotin anti-CD8a	Biolegend	100704
Biotin anti-CD11b	Biolegend	101204
Biotin anti-CD43	BD	553269
Biotin anti-DX5	Biolegend	108904
Biotin anti-Ter119	Biolegend	116204
Streptavidin Particles Plus-DM	BD	557812
FITC anti-CD44	Biolegend	103006
PE anti-CD25	Biolegend	102008
APC anti-CD62L	Biolegend	104412
PB anti-CD4	Biolegend	100428

MACS Buffer

PBS containing 0.5% BSA and 2mM EDTA