MLR (one way)

1).

Cell suspensions. (1). Remove spleen aseptically into small petri dishes containing 8ml HBSS medium. (2). Tease spleen with forceps or syringe top. (3). Wash 2x with working medium at 1200rpm for 5min. (4). Count viable cells with 0.2% Trypan blue.

2).

MLR. (1). Adjust cells to an appropriate concentration (responder cells to 3.0x106 /ml or 2.5x106 /ml and stimulator cells to 5.0x106/ml). (2). Irradiate stimulator cells with 3000R. (3). Set up cutures in 96-well round-bottomed microtiter plates with 3.0x105 or 2.5x105 and stimulator cells to 5.0x105 (200ul of each well). (4). Incubate at 370C in 5% CO2 for 3 or 4 days. (5). Pulse with 1.0uCi/well of 3H-Thymidine 16 hours before harvesting. (6). Harvest plates and determine 3H-Thymidine incorporation on scintilation counter.

3).

Working medium. RPMI 1640 with 10% FBS, 1% glutamine, 1% HEPES, 1% P/S, 1uM 2-mercaptoethanol.