COMPARISON OF MICROTITRE PLATES WITH FLAT‐BOTTOMED AND ROUND‐BOTTOMED WELLS FOR MIXED LYMPHOCYTE CULTURE (MLC)

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Abstract
To compare microtiter plates with flat-bottomed and round-bottomed wells and to standardize a method for MLC, the effects of cell number, culture time, 3H-thymidine concentration and labeling time were studied [using human blood]. On both plates, allogeneic cells induced increased RNA synthesis beginning at about 30 h and increased DNA synthesis beginning at about 50 h, if suitable cell numbers were used. On plates with flat-bottomed wells, 1.5 .times. 105 responding and stimulating cells/well had near-exponential growth on days 4 and 5, often through day 6; on plates with round-bottomed wells with corresponding cell number was 0.25-1.0 (optimally 0.5) .times. 105. Near these cell numbers, the response depended closely on the number of responding cells. On plates with flat-bottomed wells, stimulating cells had a dose-dependent effect on the response, whereas on plates with round-bottomed wells, increasing the stimulating cell dose did not consistently strengthen the response. Both plate types proved suitable for MLC experiments; plates with round-bottomed wells have the advantage of requiring smaller cell numbers. 3H-thymidine (specific activity 2000 mCi/mmol) self-suppressed its incorporation, which increased only slightly or even decreased if labeling time exceeded 12-18 h. Relative responses remained virtually unaltered with 3H-concentrations of 0.5 and 2.0 .mu.Ci/ml and with labeling times of 8 and 24 h.