Recombinant human il 3

For human leukemic cell lines, Kasumi-1, HEL, K562, Kg-1a, ME-1, and THP-1 were obtained as gifts from Dr. Ulrich Steidl lab at Albert Einstein College of Medicine and cultured in RPMI supplemented with 10% FBS and 1% penicillin & streptomycin. Human cord blood CD34⁺ cells and human primary leukemic cells were maintained in StemSpan SFEM II (STEMCELL Technologies) supplemented with primocin (100 μg/ml, InvivoGen), recombinant human TPO (100 ng/ml), recombinant human FLT3-L (100 ng/ml), recombinant human SCF (100 ng/ml), recombinant human IL-3 (20 ng/ml), and recombinant human IL-6 (20 ng/ml) (BioLegend).

Concentrations of GM-CSF, IFN-gamma, and IL-10 in cell culture supernatants were measured by ELISA, according to the manufacturer’s protocol (DuoSet Elisa, R&D Systems, Abington, UK). For measurement of IL-3, ELISA plates (NUNC Maxisorb, Thermofisher Scientific, Waltham, USA) were coated with a capture anti-IL-3 antibody (Clone P8C11; 5 µg/ml in PBS) in 100 µl/well at room temperature (RT) overnight. Plates were washed three times with PBS/0.05% Tween-20, blocked with PBS containing 1% bovine serum albumin (BSA) at RT for 1 h, and washed again with PBS. Samples were preincubated with 100 µg/ml mouse IgG1, kappa isotype control antibody (MOPC21, BioXCell, Lebanon, USA) for 1 h by RT and added to the plates for 2 h by RT (100 µl/well, diluted in PBS/1% BSA). Recombinant human IL-3 (BioLegend) was diluted from 7.8 to 500 pg/ml in PBS/1% BSA and served as standard. After washing, plates were incubated with 400 ng/ml HRP-labeled detection anti-IL-3 antibody (Clone 13) (100 µl/well) for 1.5 h at RT, and color reaction was performed with TMB Substrate Solution (BioLegend) according to the manufacturer’s protocol.