Mixed cellulose membrane

Ex vivo co-aggregation cultures of E9.5 CP and YS were performed as described (Rybtsov et al., 2014 (link)). Briefly, CPs and YSs dissected from E9.5 (23-27sp) concepti were processed into single cells as in (Swiers et al., 2013 (link)), and co-aggregated with 100,000 OP9 cells by centrifugation in 200 μL pipette tips sealed with parafilm. Co-aggregates were cultured for 7 days on top of 0.8 μm pore size mixed cellulose membranes (Millipore) at the gas-liquid interface, in Iscove’s modified Dulbecco’s medium (IMDM) (GIBCO) supplemented with 20% HyClone FCS (Fisher Scientific) and in presence of SCF, FLT3L and IL-3 (100 ng/ml, PeproTech). The culture medium was changed once after 24 hours from the beginning of the experiment. For the experiments with the gamma secretase inhibitor DAPT, 50 μM DAPT or DMSO were added to the culture media at the beginning of the experiment and after 24 hours with the media change.

CP and YS dissected from E9.0-E9.5 concepti were cultured for 1 to 7 days on top of 0.8 μm pore size mixed cellulose membranes (Millipore) at the gas-liquid interface, in Iscove’s modified Dulbecco’s medium (IMDM) (GIBCO) supplemented with 20% HyClone FCS (Fisher Scientific) and in presence of SCF, FLT3L and IL-3 (100 ng/ml, PeproTech). Explants cultured in a low oxygen environment were placed in special incubators with reduced oxygen concentration (1% O₂). For the experiments with dimethyloxalylglycine (DMOG), a range of concentrations of the inhibitor (0.1 mM, 0.5 mM, 1 mM) or DMSO were added to the culture media at the beginning of the experiment.