Histopaque gradient centrifugation

BCR-ABL-harboring human chronic myeloid leukemia cell lines K562 were from ATCC and maintained in RPMI 1640 supplemented with 10% fetal bovine serum (Life Technologies, Inc.). Imatinib-resistant, BCR-ABL T315I-harboring K562R which were derived from K562 were established and provided by Beijing Cancer Hosipital, China. Briefly, K562 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum in the presence of increasing concentration of imatinib (from 10 nM to 10 μM). The obtained single clone which harbors BCR-ABL T315I were named as K562R and routinely maintained in the same medium with 100 nM imatinib. Human embryonic kidney 293 T cells were maintained in DMEM supplemented with 10% fetal bovine serum. 100 μg/mL streptomycin and 100 U/mL penicillin were routinely added in the medium. Bone marrow of CML patients were obtained from discarded material utilized for routine laboratory tests at the Department of Hematology, Tangdu Hospital. The use of these materials is approved by the Fourth Military Medical University medical ethics committee with the informed consent obtained from the patients. Mononuclear cells and granulocytes were isolated by Histopaque gradient centrifugation (density 1.077; Sigma-Aldrich). Contaminating red cells were lysed in 0.8% ammonium chloride solution for 10 min. Isolated cells were suspended in RPMI 1640 supplemented with 15% FCS.

Peripheral lymphocytes were isolated from heparinized blood by Histopaque gradient centrifugation (Sigma, St. Louis, MO, USA). Cells lysed in ice-cold PARP buffer (including 0.4 mmol/L PMSF, 0.4 mol/L NaCl, 1% triton X-100) were collected. After incubation for 30 min on a rotator at 4°C, cell lysates were centrifuged to remove cell debris. Protein concentrations were determined with detergent-compatible reagent (Bio-Rad). PARP activity was determined using the Universal Colorimetric PARP Assay Kit (Trevigen lnc, USA) according to the manufacturer's instructions.

Data for a total of 10 patients with childhood leukemia and 6 matched healthy donors providing peripheral blood or bone marrow were obtained from The First and Seventh Affiliated Hospital of Sun Yat-sen University from 2020 to 2021. Clinical features such as age, sex, diagnosis date, disease state and white blood cell count were obtained from hospital records (Table 1).
Mononuclear cells were isolated by Histopaque gradient centrifugation (density 1.077; Sigma-Aldrich, Shanghai). Contaminating red blood cells were removed by incubation in 0.8% ammonium chloride solution for 10 min. After washing, the cells were suspended in IMDM supplemented with 10% FBS.