I'm working for a year with a murine HSC line, named FDCP-Mix. It's pain in the a** for starting in culture! Every time I want to start a new culture I spend a lot of vials and time. I follow the recommendations and culture them in IMDM, supplemented with 20% FBS, P/S and add daily, or every second day 10ng/ml mIL-3. I used to freeze them in 90% FBS, 10% DMSO, as I was used to, now I do in 70% IMDM, 20% FBS, 10% DMSO and it doesn't seem to make any difference. I freeze using Mr. Frosty and thaw at 37°C. In general I follow all instructions, apart from the one to freeze at concentration 10^6/ml - I've never frozen this diluted, I was always taught cells need to be more densely frozen, so I do around 10^7/ml (and I have more than a decade of experience in cell culturing of primary and all kinds of cell lines). Also, since viability is originally supposed to be 20% (and it almost never is that high), it'll take a lot of passages just to grow it out, which I was trying to avoid. My group has no liquid nitrogen storage tank, so we're storing everything at -80°C, which I know is not good, but for the others it seems to work out somehow... I was wondering if the L-Gln in my medium has degraded, but it was working just fine a month ago. I bought the IMDM from Biowest, with pre-added L-Gln and 25mM HEPES and once the culture is growing the cells don't seem to mind the long use of one bottle, or that the medium is expired (it literally had less than a year use time and I use very little). I made fresh medium the other day, used a fresh aliquot of IL-3 and still didn't work... I have two more vials to try, after that we'll have to buy anew and the project is almost over... I need some help...