Comparison of Microtitre Plates With Flat‐bottomed and Round‐bottomed Wells for Mixed Lymphocyte Culture (Mlc)

Abstract

To compare microtitre plates with flat‐bottomed and round‐bottomed wells and to standardize a method for mixed lymphocyte culture (MLC), the effects of cell number, culture time, ?3H‐thymidine concentration and labelling time were studied. On both plates, allogeneic cells induced increased RNA synthesis beginning at about 30 hours and increased DNA synthesis beginning at about 50 hours, if suitable cell numbers were used. On plates with flat‐bottomed wells, 1.5 × 105 responding and stimulating cells per well had near‐exponential growth on day four and five, often through day six; on plates with round‐bottomed wells the corresponding cell number was 0.25–1.0 (optimally 0.5) × 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 obvious advantage of requiring smaller cell numbers. 3H‐thymidine (spec, act 2000 mCi/mmol) self‐suppressed its incorporation, which increased only slightly or even decreased if labelling time exceeded 12–18 hours. Relative responses remained virtually unaltered, however, with 3H‐concentrations of 0.5 and 2.0 μCi/ml and with labelling times of 8 and 24 hours.

HLA Genetics of Insulin‐Dependent Juvenile‐Onset Diabetes Mellitus in Northern Finland

“…Serological typing for HLA-A, B and C antigens was done with a standard twostage microlymphocytotoxicity test. Mixed lymphocyte cultures were performed as previously reported (Herva 1977) using microtiter plates with round-bottomed wells. 0.5 x lo5 responding and irradiated (6,000 rad) stimulating cells were incubated for 5 days in 0.2ml RPMI 1640 medium, supplemented with 10% human male serum, antibiotics and glutamine.…”

Mixed Lymphocyte Culture Reactions at Delivery and in the Puerperium: Effects of Parity, Hla Antigens and Maternal Serum

“…Mononuclear cells were prepared (Bnyum 1968) as reported previously ( Herva 1976. Maternal serum for MLC inhibition and cytotoxicity testing was prepared simultaneously from clotted blood samples.…”

“…nard & Lemos 1972). However, some mothers are clearly hyporesponsive or non responsive to the cells of their infants (Ceppellini et al 1971a, Herva 1976.…”

No Significant Association between HLA and Nickel Contact Sensitivity

“…The HLA-A, B and C antigens listed in Table 1 were analyzed using the microlymphocytotoxicity method (Turunen et al 1979). The HLA-D antigens (Dwl, 2, 3, 4 and 6) were analyzed with the onedirectional mixed lymphocyte culture method (Herva 1977) using homozygous typing cells. A group of 2 17 blood donors from the same geographical area served as controls.…”