rATG-induced apoptosis in myeloma cell lines. (A) To determine the effective dose that renders apoptotic induction, different concentrations of rATG were added to myeloma cells. ARH-77 cells were isolated by Ficoll-Paque centrifugation, and 10⁵ cells/well were placed in 48-well flat-bottom plates. Cells were incubated with various concentrations of rATG (0.0001-1 μg/mL) at 37°C for 18 hours and assayed with annexin/TOPRO. Incubation of myeloma cells at increasing concentrations of rATG demonstrated a progression from live (annexin^neg TOPRO^neg) to apoptotic (annexin^pos TOPRO^neg), and finally to late apopototic/necrotic (annexin^pos TOPRO^pos) phases. (B) Concentration-dependent lysis of human myeloma cell lines. Multiple cell lines were incubated with rATG at varying concentrations for 18 hours in complement-free medium, and apoptosis was measured by annexin V binding. Data points are from n = 5 independent experiments. (C) Absence of lot-to-lot variation in apoptotic potential of rATG. MC-CAR myeloma cells were incubated with rATG for 18 hours; apoptosis was assessed by annexin V staining in 2 independent experiments for each data point. Five lots of rATG were tested.