Ficoll hypaque centrifugation

Ficoll–Hypaque centrifugation (Amersham Biosciences, Piscataway, NJ, USA) was used to isolated peripheral blood mononuclear cells (PBMCs) of SCLC patients and healthy controls. The isolated cells were resuscitated in RPMI-1640 medium (Life Technologies, Paisley, UK), supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY, USA), 1% penicillin and streptomycin (Solarbio, Beijing, China). Co-cultured with recombinant human IL-2 (10 ng/mL, R&D Systems, Minneapolis, MN, USA), anti-human CD3 antibody (1 ng/mL; eBioscience, San Diego, CA, USA) and anti-human CD28 antibody (1 ng/mL; eBioscience).

Peripheral blood mononuclear cells (PBMCs) from the patients and healthy controls were isolated from heparinized venous peripheral blood using Ficoll–Hypaque centrifugation (Amersham Biosciences, Piscataway, NJ, USA). Isolation of circulating CD4⁺ T cells was performed using anti‐CD4‐coated magnetic beads and MS column (Miltenyi Biotec, Bergisch Gladbach, Germany) separation. The purity of the isolated cells was found to be >90% by flow cytometry.
PBMCs or CD4⁺ T cells were resuspended in RPMI‐1640 medium (Life Technologies, Paisley, UK) supplemented with 10% heat‐inactivated fetal bovine serum (Gibco, Grand Island, NY, USA), 1% penicillin and streptomycin (Solarbio, Beijing, China), and recombinant human IL‐2 (5 ng/mL, R&D Systems, Minneapolis, MN, USA), anti‐human CD3 antibodies (1 ng/mL; eBioscience, San Diego, CA, USA), and anti‐human CD28 antibodies (1 ng/mL; eBioscience). The cells were then seeded on 24‐well plates.
Indirubin was dissolved in dimethyl sulfoxide (DMSO, Sigma‐Aldrich) to generate a stock solution of 5 mg/mL. Cultures of PBMCs or CD4⁺ T cells were treated with indirubin at a concentration of 0.01, 0.1, 0.2, 1, 2, and 10 µM, whereas 1‰ DMSO was used as vehicle controls. After 72 hours of incubation with indirubin or DMSO, PBMCs or CD4⁺ T cells were collected for flow cytometry, RNA extraction, and western blotting.

Human HL-60 (ACC 3, FAB M2) and KG-1 (ACC14, relapse) AML cell lines were obtained from DSMZ (Leibnitz Institute, German Collection of Microorganisms and Cell Cultures; Leibnitz; Germany). Primary AML cells were obtained as mononuclear cells separated by Ficoll–Hypaque centrifugation (Amersham, Buckinghamshire, UK) from 3 newly diagnosed AML patients (1. 46 XX, M1; 2. 46 XY, M2; 46 XY, del9(q21;q34), M2; karyotype and Fab, respectively) after informed consent signature (local ethics committee approval code: 147/2013/O/Tess; approved 11 June 2013). The cells were cultured in RPMI 1640 medium (Lonza, Milan, Italy), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich, St. Louis, MO, USA), 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin (MP Biomedicals, Milano, Italy) (complete RPMI) and maintained at 37 °C and 5% CO₂. CD3⁺ and CD14⁺ cells were purified by magnetic separation (MiltenyiBiotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions from mononuclear cells separated from buffy coats of healthy donors by Ficoll–Hypaque centrifugation (Amersham; Burlington; Canada) after informed consent signature (local ethics committee approval code 94/2016/O/TES). Purity of cell populations was always >90%.