Autophagy Inhibitor, 3-MA

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	rest cells for 2 h and then treat with absence or presence of the autophagy inhibitor (5 mM, 3-MA)

Protocol tips
rest cells for 2 h and then treat with absence or presence of the autophagy inhibitor (5 mM, 3-MA)

Publication protocol

Human lymphoblastic leukemia cell line REH (ATCC CRL-8286) was obtained from American Type Culture Collection (Manassas, VA), while human promyelocytic leukemia cell line HL-60 (ECACC 98070106) was purchased from the European Collection of Animal Cell Cultures (Salisbury, UK). Peripheral blood mononuclear cells (PBMC) were obtained from venous blood of three patients with blastic transformation as the first presentation of the chronic myeloid leukemia (CML), with more than 108/l WBC count and myeloblast/promyelocyte count > 50%. The diagnosis was established at the Outpatient Clinic of the Outpatient & Diagnostic Department, Clinic of Hematology, Clinical Centre of Serbia (Serbia, Belgrade), according to the diagnostic criteria for classification of tumors of hematopoietic and lymphoid tissue [24]. Control PBMC were obtained from three healthy volunteers, age- and sex-matched with leukemic patients. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethical Committee of the Clinical Centre of Serbia and the Ethical Committee of the School of Medicine, University of Belgrade. Each volunteer provided a written consent for participation in the study after being informed about all the details of the study. All patients provided two informed written consents, one general concerning diagnostic procedures, and another one concerning the scientific analysis, because the samples were taken during regular diagnostic workup. Blood draws were conducted with syringes containing 10% (v/v) of 3.8% sodium citrate as an anticoagulant. PBMC were isolated by density gradient centrifugation using LymphoPrep (Axis Shield, Oslo, Norway) and immediately used for experiments. The cell lines and PBMC were incubated at 37°C in a humidified atmosphere with 5% CO2, in a HEPES (20 mM)-buffered RPMI 1640 cell culture medium supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate 10 ml/l penicillin/streptomycin (all from Sigma-Aldrich, St. Louis, MO). Cells were incubated in 96-well flat-bottom plates (4×104 or 1.2×105 cells/well for the cell lines or PBMC, respectively) for the viability assessment, 24-well plates (3×105 cells/well for the cell lines) for the flow cytometry analysis or in 100 mm cell culture dishes (2.5×107 or 1×108 cells/well for the cell lines or PBMC, respectively) for the immunoblotting and electron microscopy. Cells were rested for 2 h and then treated with cytarabine in the absence or presence of the autophagy inhibitors bafilomycin A1, chloroquine and 3-methyladenine, or mTOR activator leucine (all from Sigma-Aldrich), as described in Results and Figure legends.

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