Nonadherent cells were harvested from (FVB/N) Fancd2+/+, Fancd2+/− and Fancd2−/− mouse LTBMC at week 4 and cultured in six-well tissue culture plates in Iscove's modified Eagles medium (IMDM) supplemented with 20% fetal calf serum (FBS) and 1.0 ng/mL Interleukin 3 (IL-3) (Peprotech, Rocky Hill, NJ). The Fancd2+/+, Fancd2+/− and Fancd2−/− cell lines were passaged weekly for 10 weeks to establish primary IL-3-dependent cell lines (16 (link)).
Clonal cell sublines were established from each of the (FVB/N) Fancd2+/+, Fancd2+/− and Fancd2−/− parent lines by expansion of single colonies. Cells from primary IL-3-dependent cell lines were plated in 0.8% methylcellulose supplemented with 10% IMDM, 30% fetal bovine serum (FBS), 1% bovine serum albumin, 2 ng/mL IL-3 (Stemcell Technologies, Vancouver, Canada) at variable cell densities. At day 14, individual colonies were harvested and each cultured in a well of a 96-well plate in 0.2 mL of IMDM supplemented with 30% FBS and 1 ng/mL IL-3. Cells were then replated in methylcellulose-containing medium, colonies selected at day 14 and cultured as above to establish subcloned lines. Confirmation of genotype after repeated subcloning was performed for each cell line as published previously (16 (link)).
Clonal cell sublines were established from each of the (FVB/N) Fancd2+/+, Fancd2+/− and Fancd2−/− parent lines by expansion of single colonies. Cells from primary IL-3-dependent cell lines were plated in 0.8% methylcellulose supplemented with 10% IMDM, 30% fetal bovine serum (FBS), 1% bovine serum albumin, 2 ng/mL IL-3 (Stemcell Technologies, Vancouver, Canada) at variable cell densities. At day 14, individual colonies were harvested and each cultured in a well of a 96-well plate in 0.2 mL of IMDM supplemented with 30% FBS and 1 ng/mL IL-3. Cells were then replated in methylcellulose-containing medium, colonies selected at day 14 and cultured as above to establish subcloned lines. Confirmation of genotype after repeated subcloning was performed for each cell line as published previously (16 (link)).
