Flat bottom tissue culture plates

For ³H thymidine incorporation, freshly isolated human peripheral blood mononuclear cells (MNC; 1 × 10⁶/mL cells) were cultured in flat-bottom tissue culture plates (Sarstedt, Newton, MA, USA) and stimulated with 0.1 ng/mL wild-type recombinant TSST-1 (toxin) for four days in a humidified atmosphere. Negative serum was taken from a human unvaccinated donor, which was not included in the study. Phytohemagglutinin was used as a positive control.
Induction of proliferation was quantified by counting incorporated [³H] thymidine after incubation for 4 days in a humidified atmosphere (37 °C, 5% CO₂). On the fourth day, 0.5 µCi/well ³H-thymidine (ICN, Irvine, USA) was added, suspensions were incubated for 18 h and plates were frozen at −20 °C overnight. Incorporated radioactivity was counted with a Microbeta Trilux 1450 scintillation counter (Wallac, Turku, Finland). Counts per minute were given as corrected counts per minute (ccpm), based on automated counter protocols defining counting parameters. Neutralization of T cell activation was defined as a ≥50% decrease of thymidine incorporation compared to results for negative serum.

Human T cells were purified from hPBMCs by non-T cell depletion using the Pan T cell isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). Purity of human T cells usually exceeded 97%. All cells were cultured at 37°C with 5% CO₂ in 24-well (1x10⁶ cells in 1 ml medium) flat-bottom tissue culture plates (Sarstedt, Nümbrecht, Germany) using X-VIVO 15 medium (Lonza, Basel, Switzerland). 24-well flat-bottom tissue culture plates (Sarstedt, Nümbrecht, Germany) were coated before with 5 μg/ml OKT3 (Janssen-Cilag, Neuss, Germany) in 200 μl PBS (Biochrom, Berlin, Germany) per well at 4°C for 24 h. Plates were washed twice with 400 μl PBS to remove unbound antibody prior to addition of human T cells in 1 ml X-VIVO 15 medium. Human T cells cultured on PBS-treated wells without antibody were used as negative control. CD3 expression on human T cells was measured by a flow cytometric analysis.