BJAB, Raji, Ramos, Daudi, and Jurkat cells were purchased from ATCC and grown in RPMI medium with 10% FBS (HyClone) in a 5% CO2 atmosphere at 37°C, and split 2–3 times per week. Cell lines were authenticated by STR analysis (MD Anderson Cancer Center Characterized Cell Line Core) and regularly tested for mycoplasma (Lonza).
For drug treatment, 0.5e6 cells/mL in RPMI + 5% FBS were seeded into 24-well plates and treated with indicated doses of FasL (Enzo) or edelfosine (Sigma-Aldrich) for 20 hours, doxorubicin (Sigma-Aldrich) for 48 h, or 10 ng/mL of super FasL (sFasL; Enzo) for 16 h.
BJAB cells (1e6 cells/mL) were incubated with 50 μg/mL of milatuzumab (humanized anti-CD74 antibody, hLL1; Immunomedics, Inc.) for 10 min prior to addition of 20 μg/mL of goat anti-mouse or goat anti-human IgG (both from Jackson ImmunoResearch). Cells were washed after 30 min and subsequently incubated with either 50 ng/mL of anti-Fas antibody CH-11 (Millipore) or recombinant FasL (Enzo). Cells were harvested 24 h later and analyzed for apoptosis by propidium iodide staining and flow cytometry, as described previously [27 (link)].
For drug treatment, 0.5e6 cells/mL in RPMI + 5% FBS were seeded into 24-well plates and treated with indicated doses of FasL (Enzo) or edelfosine (Sigma-Aldrich) for 20 hours, doxorubicin (Sigma-Aldrich) for 48 h, or 10 ng/mL of super FasL (sFasL; Enzo) for 16 h.
BJAB cells (1e6 cells/mL) were incubated with 50 μg/mL of milatuzumab (humanized anti-CD74 antibody, hLL1; Immunomedics, Inc.) for 10 min prior to addition of 20 μg/mL of goat anti-mouse or goat anti-human IgG (both from Jackson ImmunoResearch). Cells were washed after 30 min and subsequently incubated with either 50 ng/mL of anti-Fas antibody CH-11 (Millipore) or recombinant FasL (Enzo). Cells were harvested 24 h later and analyzed for apoptosis by propidium iodide staining and flow cytometry, as described previously [27 (link)].