Lc3b antibody kit for autophagy

To determine the cytotoxicity induced by AraC, Aza, Gal-9, and CQ (from the LC3B Antibody Kit for Autophagy, L10382, Invitrogen^TM, Carlsbad, CA, USA), 5 × 10⁴ cells (either cell lines or primary patient-derived material) were plated in a 48 well plate with 200 µL RPMI supplemented with 10% FCS (for cell lines) or Gartner’s medium (for patient-derived AML cells or CB cells) and treated with the indicated concentrations of Gal-9, AraC, Aza or CQ. In the case of combinational treatments, Gal-9 and AraC were added simultaneously, whereas cells were pre-incubated with Aza for 16 h before adding Gal-9. For experiments with Z-VAD-fmk (R&D Systems, Inc., FMK001, Wiesbaden, Germany), cells were pre-treated with 20 µM Z-VAD-fmk for 16 h and again treated with 20 µM Z-VAD-fmk freshly added 1 h before adding Gal-9 or Staurosporine (Sigma Aldrich, St. Louis, MO, USA). For long-term assays using patient-derived AML cells (5–7 days), wells were first coated with MS5 cells (pre-plated 1 day before the assay to reach confluency at the day of use) before AML cells were layered on top of the stromal layer. For the CQ assays, cells were incubated on top of MS5 cells. Cytotoxicity was assessed using either flow cytometry-based cell counts, Annexin-V staining, DioC6 staining, or MTS assay.