Donor peripheral blood mononuclear cells (PBMCs) are cultured with recipient PBMCs. The cells are photodepleted following treatment with 4,5-dibromorhodamine methyl ester (TH9402).
Peripheral blood mononuclear cells (PBMCs) are isolated using density-gradient centrifugation. Donor PBMCs are cultured with 20 Gy-irradiated recipient PBMCs in T-75 culture flasks (ratios 1:1 or 2:1) in RPMI-1640 medium containing 10% donor defibrinated plasma (cell concentration of responder cells: 3–5 × 106/ml; thickness: 0.5 cm; volume: 37.5 ml for each flask). Cells are then treated with 4,5-dibromorhodamine methyl ester (TH9402, 5 μM). When TH9402 incorporation, measured by flow cytometry, is observed in 20,000-25,000 cells, cells are exposed to a fluorescent light-emitting device (0.1 J/cm2, 514 nm). Treated cells are cultured for 3 days to completely eliminate TH9402.