The indicated ALL cells were plated at a density of 1×106 cells per mL in different conditions in an irradiated OP9 bone marrow stroma cells [16 (link)–17 ]. OP9 cells were purchase from ATCC. Coculture of human ALL cells with OP9 cells was in MEM-α medium supplemented with 20% FBS (Sigma), 1% L-glutamine (Gibco), 1% penicillin/streptomycin (Hyclone). The rate of proliferation and viability was monitored every other day by manually counting the viable cells and total cells using Trypan Blue stain (Sigma). For the E670-based proliferation assays, 5 × 106 ALL cells were labeled with 1 μm E670 (Invitrogen) and cultured for 7 days. E670 dilution was measured by gating on live E670+ cells using flow cytometry (Accuri C6).
For testing drug resistance in vitro, 1×106 cells (PT-1, PT-2, or PT-3) per mL were cultured in different conditions in an irradiated OP9 bone marrow stroma cells. Vincristine (2.5 nM) or nilotinib (300 nM) were added every other day. Non-adherent leukemia cells were carefully collected from the stromal layer. Viability of the ALL cells was determined by excluding Trypan blue-positive ALL cells.
For testing drug resistance in vitro, 1×106 cells (PT-1, PT-2, or PT-3) per mL were cultured in different conditions in an irradiated OP9 bone marrow stroma cells. Vincristine (2.5 nM) or nilotinib (300 nM) were added every other day. Non-adherent leukemia cells were carefully collected from the stromal layer. Viability of the ALL cells was determined by excluding Trypan blue-positive ALL cells.
