Methyl 3h thymidine 3htdr

To detect T‐cell responses, PBMC (1 × 10⁵) were cultured with Aspergillus fumigatus extract (Allergon, Angelholm, Sweden), tetanus‐toxoid (Statens Serum Institute, Denmark) and the MHC Class II peptide mix incorporating peptides from CMV, EBV, influenza and tetanus (CMV‐EBV‐Flu‐Tet peptide pool; JPT Peptide Technologies, Berlin, Germany). Phytohaemagglutinin (PHA; Sigma, MO, USA) was included as the positive control and PBMC alone in the absence of antigen included to assess background levels of proliferation. After 4 days 30 μL of supernatant was removed from each culture and stored at −80°C for cytokine analysis. The PBMC cultures were then supplemented with 30 μL fresh media containing 1 μCi of methyl‐³H Thymidine (³HTdR; MP Biomedicals, Australia) and harvested 16–20 h later. Incorporation of ³HTdR was measured by liquid scintillation spectroscopy as counts per minute (cpm), and mean counts per minute with standard deviation (SD) was calculated for triplicate cultures (SD < 20% for all experiments). Results are expressed as Stimulation Index (SI) which is the ratio of cpm for proliferation in presence of antigen/proliferation in PBMC alone culture and an SI > 2.5 was defined as a positive antigen‐specific response.

Cultures performed in 96-well U-bottom plates were pulsed with 0.25 μCi of methyl ³H-thymidine ([³H]TdR; MP Biomedicals, Irvine, CA, United States) for 6 h. DNA was then harvested onto glass fiber filter mats using a Titer-Tek cell harvester (Skatron Instruments, Lier, Norway). [³H]TdR incorporation, which is a measure of DNA synthesis, was determined using a Beckman LS6000IC liquid scintillation counter (Beckman Coulter, Inc., Brea, CA, United States).