Methyl 3h thymidine

Methyl-[³H]-thymidine was purchased from ICN Biomedicals, Inc. (Irvine, CA); ²²NaCl and ⁸⁶Rb were obtained from Perkin Elmer (Waltham, MA) and Isotope (Russia). Marinobufagenin was kindly provided by Dr. A. Y. Bagrov (NIH, Baltimore). The remaining chemicals were from Gibco BRL (Gaithersburg, MD), Calbiochem (La Jolla, CA), Sigma (St. Louis, MO), and Anachemia (Montreal, QC).

A total of 5 × 10⁴ PBMC/well were cultured for 1 h at 37°C and 5% CO₂ in complete medium in the absence or presence of different peptide concentrations (as indicated in each figure) in 96-well flat-bottomed microplates (Linbro, Aliso Viejo, CA, USA). In some experiments, PBMC were also stimulated with 2 μg/mL PHA-P or 25 ng/mL PMA for 48 h or 72 h with or without addition of peptides. Afterwards, 0.5 μCi/well [methyl-3H]-thymidine (ICN Biomedicals, Inc., Irvine, CA, USA) was added to cultures for the last 18 h. Cells were then harvested (PHD Harvester, Cambridge Tech) in glass fiber strips (Cambridge Technology, Watertown, MA, USA) and assayed for [methyl-3H]-thymidine incorporation by liquid scintillation counting β-scintillation system Beckman LS 6500 (Beckman Instruments, Fullertown, CA, USA).
Peptides-treated or peptides-untreated Jurkat cells (5 × 10⁴) were stimulated with 2 μg/mL ionomycin or 2 μg/mL PHA-P and processed as described above for primary cells. The stimulation index percentage (SIP) was defined as being the ratio of mean [methyl-3H]-thymidine (counts per minute) incorporated in the presence of a peptide to that incorporated in the absence of peptide (×100).