Generation of Hoxb8 neutrophil progenitor cells

HoxB8 cells were generated as previously described [22]. In brief, bone marrow cells from C57Bl/6 mice were stimulated in Roswell Park Memorial Institute supplemented with recombinant mouse cytokines IL-3 (10 ng/ml), IL-6 (20 ng/ml), and 1% SCF supernatant from SCF secreting CHO cells and cultured for 3 days. Cells were infected with retrovirus expressing ER-Hoxb8 fusion, which is active in the presence of estradiol and grown in media containing β-estradiol for 2 weeks to ensure the outgrowth of transformed progenitors. Hoxb8 positive neutrophil progenitors were differentiated by washing out β-estradiol 2–3× with warm phosphate-buffered saline (PBS). To differentiate, cells were seeded at 1 × 10⁵cells/ml in 3 ml differentiation medium (Optimem Glutamax, 10% FCS, 1% Pen/Strep, 1% SCF supernatant) in a six-well plate and cultured for 4 days.

To generate C/EBPα-overexpressing cells, HoxB8 progenitors were infected with pMIG retroviruses expressing either wild type or the indicated mutants of C/EBPα. As pMIG contains an IRES-GFP, cells were sorted for GFP+ populations to select for infected cells. For each construct, multiple infections were performed at different times to avoid clonal selection.