RPMI 1648 medium (Gibco, Karlsruhe) was supplemented with 25 mM HEPES buffer 1 mM l-glutamine (Gibco, Karlsruhe), 1× Penicillin/Streptomycin (Cölbe, find more 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 Linsitinib mw 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 Carnitine palmitoyltransferase II 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.