RPMI 1648 medium (Gibco, Karlsruhe) was supplemented with 25 mM HEPES buffer 1 mM l-glutamine (Gibco, Karlsruhe), 1× Penicillin/Streptomycin (Cölbe, selleck chemical PAA) and 10% heat-inactivated fetal calf serum. 8 ml were aliquoted into 50 ml polypropylene tubes (Sarstedt, Nürnbrecht) and warmed to 37 °C. Upon removal from liquid nitrogen storage, no more

than two cryovials at a time were thawed in a 37 °C water bath until the cell suspension was melting and a little ice remained. One ml of warmed media was slowly added to the thawed PBMC and the cell suspension had been transferred to a corresponding polypropylene tube (final volume 10 ml). The tubes were centrifuged at 400 g for 5 min at room temperature. The PBMC were resuspended in 10 ml medium per 1 × 107 cells and transferred in a cell incubator (5% CO2, 37 °C) overnight with the cap of the tube loose. The effect of the 3 different storage conditions on cell recovery was evaluated using the ViCell cell analyser (Beckman Coulter,

Krefeld). Five samples per donor Mitomycin C order per storage condition were thawed and cell recovery and viability measured immediately post-thaw and again after overnight culture using the trypan blue dye exclusion test. Each sample was measured three times. Recovery (%) (after thawing): %recovery=(number of viable PBMC after thawing/number of frozen viable PBMC)×100 Recovery (%) (after overnight culture): %recovery=[number of viable PBMC after overnight rest/(number of frozen viable PBMC-number of viable PBMC removed for measurement directly after thawing)]×100 Viability: %viability=(number of viable PBMC/number of total PBMC)×100 PBMC were assayed for IFN-γ production in the presence of CMV pp65 peptide pool (BD Bioscience,

Heidelberg), CEF peptide pool (CTL, Bonn), PHA (Sigma–Aldrich, Taufkirchen) and background control (culture Progesterone media containing 0.4% DMSO) in triplicates. 96 well plate anti-human-IFN-γ mAb 1-D1k precoated (Mabtech, Hamburg) were washed four times with PBS (Gibco, Karlsruhe) and blocked with culture medium, RPMI 1648 medium (Gibco, Karlsruhe) containing 25 mM HEPES buffer 1 mM l-glutamine (Gibco, Karlsruhe), 1× Penicillin/Streptomycin (Cölbe, PAA) and 10% heat-inactivated fetal calf serum, for 30 min. Cryopreserved PBMC were thawed as described above and used the next day. Approximately 1 × 105 PBMC were added to the CEF, CMV and background wells and 0.5 × 105 PBMCs to the PHA wells. CEF peptides and CMV peptides were added to a final concentration of 2 μg/ml/peptide and 1.75 μg/ml/peptide, respectively. The final PHA concentration was 4 μg/ml. The final DMSO concentration was between 0.1% and 0.25%. The plates were incubated at 37 °C, 5% CO2 for 20–22 h.