Methocult h4034

To analyze the effect of the overexpression or knockdown of Pot1a on colony-forming activity, Pot1a- or shPot1a-transduced LSK cells were cultured for 1–4 weeks. After in vitro culture, GFP⁺ LSK cells were sorted and cultured in MethoCult™ GF M3434 medium for mouse cells and MethoCult™ H4034 for human cells (Stemcell Technologies).
To analyze the effect of MTM-POT1a on the maintenance of HSC colony formation activity, LSKCD41⁻CD48⁻CD150⁺ cells were cultured for 10 days in SF-O3 medium in the presence of 0.1% BSA, 100 ng ml^–1 SCF, and 100 ng ml^–1 TPO with or without MTM-POT1a. Lin⁻ cells were sorted and their ability to form colonies was then assessed. To analyze the effect of MTM-POT1a on the maintenance of HSC numbers, LT-HSCs (LSKCD41⁻CD48⁻CD150⁺ cells) were cultured for 3 weeks in SF-O3 medium in the presence of 0.1% BSA, 100 ng ml^–1 SCF, and 100 ng ml^–1 TPO with or without MTM-POT1a. After culture, the number of LSK cells and LT-HSCs was examined. For examination of colony formation in hCB HSCs after in vitro culture, Lin⁻CD34⁺CD38⁻CD45RA⁻CD90⁺CD49f⁺ cells were cultured for 10 days in StemSpan SFEM (Stemcell Technologies) in the presence of 100 ng ml^–1 SCF, 100 ng ml^–1 rhFL, and 20 ng ml^–1 TPO with or without MTM-POT1. After culture, Lin⁻ cells were sorted and cultured in MethoCult™ H4034 medium.

The suspended L-428 cells (number = 2,500)were plated in culture dishes with 1 ml of semisolid methylcellulose culture medium (Methocult H4034; Stem Cell Technologies, Meylan, France) in the presence or absence of DPN 10 nM. Duplicates aliquots (1 mL) were plated in 35-mm Petri plates and incubated further at 37°C. Colonies were counted at 10 days.