Analysis of CD30 redox state by flow cytometry
For reduction of cell surface CD30, 2.5 × 105 cells were incubated with 5 μM Trx1 together with 200 μM DTT or 100 nM human Trx reductase (TrxR)/500 μM NADPH for 30 min at 4°C. To monitor reduction of cell surface CD30, cells were stained with anti-human CD30 monoclonal antibody MAB229 (R&D Systems), anti-human CD30 monoclonal antibody Ki-1 (Santa Cruz) or anti-human CD30 monoclonal antibody Ber-H2 (DakoCytomation) followed by incubation with R-PE-conjugated goat F(ab')2 anti-mouse Ig's (Biosource). For control, cells were stained with R-PE-conjugated anti-human CD28 monoclonal antibody (BD Pharmingen). Cells were analyzed using a FACSCalibur (Becton Dickinson) and CellQuest software.