Methocult gf m3434 methylcellulose

For clonogenic assays, Lin- cells were plated in Methocult GF M3434 methylcellulose (03444, Stem Cell Technologies) and cultured. The BET bromodomain inhibitor (+)-JQ1 was added to a final concentration of 50 nM and hematopoietic colonies were scored after 7 days of culture at 37°C and 5% CO₂.
For clonogenic assays using peripheral blood derived cells, mononuclear cells were isolated by lysing the whole blood with ACK lysis buffer (10-548E, Lonza) and incubating during 5 min at 37 °C, cells were washed with PBS and the process repeated if the cell pellet looks red. 300, 000 total mononuclear cells were plated in Methocult GF M3434 methylcellulose (03444, Stem Cell Technologies) during 14 days at 37 °C and 5% CO₂. Pictures were taken with the STEMvision System (StemCell Technologies).

For clonogenic assays,20,000 Lin⁻ cells per condition (in triplicate) and 2000 sorted LSK cells per condition (in triplicate) were plated in Methocult GF M3434 methylcellulose (03444,Stem Cell Technologies) and cultured for 7 days. The TGFβ inhibitors such as AVID200 (Forbius), SD208 (S7071, Sigma-Aldrich) and 1D11 (Genzyme) were added in graded concentrations in the cultures. For acetaldehyde treatments, bone marrow cells were exposed to increasing doses of acetaldehyde (00070–100ML, Sigma-Aldrich) for 4 h, washed and then cultured in Methocult GF M3434 methylcellulose. The hematopoietic colonies were scored after 7 days of culture at 37 °C and 5% CO₂.
For proliferation assays, sorted LSK cells were cultured in serum-free medium StemSpan SFEM (09600, StemCell Technologies) containing 1% penicillin/streptomycin (15140–122, GIBCO), 2% L-glutamine (25030–081GIBCO), 100 ng/ml TPO (315–14-10UG, Peprotech) and 100 ng/ml SCF (250–03-10UG, Peprotech). After 48 h in culture, DNA damage was assessed by immunofluorescence staining the cells to detect γH2AX foci formation (2577s, Cell Signaling) as described [13 (link)].