The erythrocytes in the cell mixture were
washed via hyperosmotic haemolysis,and the cells were resus-
pended to a final density of 5106 cells/ml in RPMI 1640 medium
supplemented with 10 % newborn bovine serum(Sangon Biotech,
China), 100 mg/ml streptomycin,and 100 U/ml penicillin.Spleen
cells (100 μl/well) were seeded into a 96-well plate containing
ConA (8 μg/ml), then cultured for 3 days in 5 % CO2 atmosphere at
37 1C, and further incubated for 4.5 h with 10 μl MTT(5 mg/ml)
per well.The plate was then centrifuged at 200 g for 15 min,the
resulting supernate was discarded.A volume of 100 μl dimethyl
sulfoxide(DMSO)was added to each well,which was then shaken
until all crystals dissolved.The absorbance at 570 nm was mea-
sured on a microplate reader(ELX800,USA).