Methocult h4434 methylcellulose medium

Transfected cells were plated in 96-well plates at a density of 5 × 10³ cells/well in quintuplicate. Ten microliters of a WST-1 working solution (Keygen, Nanjing, China) was added to each well, and the cells were incubated for 2 hours. The absorbance at 450 nm was measured using a microplate reader. The inhibition rate of cell proliferation was calculated as follows: % of inhibition rate = each time point (OD^{treated well} − OD^{blank well})/original point (OD^{unreated well} − OD^{blank well}). Cell apoptosis was analyzed using FACS and Annexin V–APC. For cell-cycle analysis, 10⁵ cells were washed with cold PBS, fixed in 70% ethanol, washed with PBS, and re-suspended in 1 mL of 7-AAD staining reagent (50 mg/ml propidium iodide and 1 mg/ml RNAse). Samples were incubated in the dark for 30 min before cell cycle analysis. Cell cycle was measured using FACS Calibur. The percentages of cells in the G1, S and G2 phases were calculated using Cellquest software. For colony formation, transfected cells were plated in 12-well plates in Methocult H4434 methylcellulose medium containing SCF, GM-CSF, IL-3, and erythropoietin (StemCell Technologies, Hangzhou, CN) at 2 × 10³ cells/well in duplicate wells for each condition. Cells were incubated for 14 days in a humidified incubator at 37 °C, and colonies that contained at least 30 cells were counted.