Preparation of Human Lymphocytes

C RPMI medium, 5% ECS (v/v), 0.35ml/100ml 2-mercapto-ethanol, lOU/lOOml streptomycin, lOU/lOOml penicillin, 29 mg/100 ml L-glutamine 

Mix 10 ml freshly collected venous peripheral blood intensively with 2.5 ml Soln. A at RT. Then add 12.5 ml Soln. B, warmed to RT. Carefully cover the top of 25 ml of Lymphoprep (p = 1.077 g/ml) with the diluted blood and spin with 800 x g (about 1600 rpm) at 20 °C for 30 min. 

Discard the supernatant and harvest the cells. Dilute the cell suspension with the same volume of Soln. B and centrifuge at 20 °C with 500 X g (1000 rpm) for 10 min. Dilute the sediment with 30 ml of Soln. B and spin again. 

Suspend the sediment in 10 ml of Soln. C and centrifuge at 4 °C with 1500 rpm for 5 min. Repeat this step until the lymphocytes are free from thrombocytes. Dilute the lymphocyte concentrate with about 20 ml Soln. D for further experiments. 

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The regulation of PRA synthesis from PRPP and glutamine (reaction 1) and PRPP and anmonia (reaction 2) was examined in cell-free preparations of human lymphocytes. ... 

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