Alexa fluor 647 mouse anti human cd138 antibody

Fresh de-identified primary MM patient BM samples were obtained from the UCSF Hematologic Malignancies Tissue Bank in accordance with the UCSF Committee on Human Research-approved protocols and the Declaration of Helsinki. BM mononuclear cells were isolated by density gradient centrifugation with Histopaque-1077 (Sigma Aldrich), and washed with 10 mL D-PBS 3 times. Mononuclear cells were resuspended in a small volume (~1.5 mL) of media (RPMI1640, 10% FBS, 1% penicillin/streptomycin, 2 mM glutamine) and incubated at 37 °C, 5% CO₂ for 15 min. Isolated mononuclear cells from MM patient BM were adjusted to 2 E+5 cells per well in a 96-well plate. Cells were stimulated with 50 ng mL⁻¹ recombinant human IL-6 (ProSpec) for 17 h before treatment with E7107 or DMSO for 24 h. Cells were then stained with 10 μL Alexa-Fluor 647 mouse anti-human CD138 antibody (BD Pharmingen, cat# 562097; RRID:AB_10895974) or Alexa-Fluor 647 IgG κ isotype (BD Pharmingen, cat# 557714; RRID:AB_396823) control and 2 μL SyTOX Green (Thermo, S34860) per 1 mL FACS buffer (D-PBS, 5% FBS). Resuspended cells are characterized with a CytoFLEX fluorescence cytometer (BD).