Colony Forming Assay for Murine and Human Hematopoietic Cells

Methocult M3434 (murine) and H4435 (human) from STEMCELL Technologies were used for colony-forming assays. Methylcellulose was supplied with the indicated concentration of Epag or rTPO before cell seeding. Mouse bone marrow cells after red blood lysis were seeded at the concentration of 30,000 cells/mL. Bone marrow mononuclear cells from healthy donors and patients were seeded at the concentration of 100,000 cells/mL. CD34⁺ cells were infected with lentiviral TET2 targeting shRNA or nontargeting scr shRNA as described and characterized in our previous study (31 (link)). Two days after lentivirus infection, CD34⁺ cells were seeded at the concentration of 5000 cells/mL in Methocult in the presence of 5 μg/mL puromycin. Colonies were scored and cells were harvested for replating or flow cytometry analysis by FACSVerse (BD Biosciences) on days 10 to 14.