A microplate culture system has been used to standardise mouse lymph node lymphocyte responses to concanavalin A (Con A), phytohaemagglutinin (PHA), pokeweed mitogen (PMW), endotoxin (LPS) and allogeneic lymphocytes and optimum culture and [3H]thymidine (3H-Tdr) labelling conditions determined. The effects on lymphocyte transformation of microplate well-shape, foetal calf serum, cell concentration, mitogen dose, culture time, buffering system and initial pH of culture were examined. These factors showed different effects, both quantitative and qualitative, on the kinetics of the responses to the various stimulants resulting in dissimilar optimal culture conditions. This probably reflects either the different subpopulations of lymphocytes activated by these stimulants, or the different modes of action of the stimulants. Optimal 3H-Tdr labelling conditions were achieved with saturation concentrations of exogenous Tdr of at least 3.0 μg/ml during culture in the most highly proliferating cultures. At saturation concentrations, any specific activity (SA) of 3H-Tdr could be used for quantitation since lymphocytes in different states of activation were affected by the radiobiological effects to the same extent. However, it was calculated from the parabolic relationship of 3H-Tdr incorporation and SA that at saturation concentrations the maximal SA that should be used to provide the highest uptake is 1.3 Ci/mM for a 24 hr pulse.
ASJC Scopus subject areas
- Immunology and Allergy