Methocult h4035 optimum without epo

MOLM-13, OCI-AML3, or MV4-11 (156 cells per condition) were plated with DMSO or 1 µM EPZ in MethoCult H4035 Optimum Without EPO (StemCell Technologies, Cambridge, MA, USA) and incubated at 37 °C with 5% CO₂ for 7 days. Images were taken with the STEMVision Instrument, and the colonies were counted manually. For primary AML samples, cells were plated at the appropriate density (5 × 10⁴–2 × 10⁵ cells/well) with DMSO or 1 µM EPZ in MethoCult H4035 Optimum Without EPO and incubated at 37 °C with 5% CO₂ and 1% O₂ for 14 days. Images were taken with the STEMVision Instrument, and colonies were counted using the appropriate STEMVision algorithm or manually by two counters blinded to experimental conditions on the Revolve Microscope (ECHO). Samples that produced fewer than 20 colonies counted in the DMSO condition were excluded. For the normal donor CD34+ CFU assays, normal bone marrow was obtained from AllCells. The Human CD34 MicroBead Kit (Miltenyi Biotec, Gaithersburg, MD, USA) was used to isolate CD34+ cells. A total of 1000 CD34+ cells/well were plated with DMSO or 1 µM EPZ in MethoCult H4035 Optimum Without EPO and incubated at 37 °C with 5% CO₂ and 1% O₂ for 7–10 days. Colonies were counted by two counters blinded to the experimental conditions using the Revolve Microscope (ECHO). Values are reported as averages of the two separate counters.