8 well chambers

Cells were cultured on 8-well chambers (BD Biosciences) and fixed with 4% paraformaldehyde/PBS for 15 min at room temperature. For 3D cultures cells were seeded (3 × 10³/well) in quadruplicate onto Matrigel (BD Biosciences) beds in 8-well culture slides to prepare three-dimensional cultures as described earlier [61]. The media was changed every 48 h for 8 consecutive days. Colony morphology was determined by phase-contrast microscopy. For immunostaining, cells were permeabilized with 0.5% Triton X-100/PBS and blocked with 10% normal goat serum (Invitrogen) in PBS for 1 h. Primary antibodies were incubated overnight at 4°C in blocking buffer and washed 3 times with washing buffer (0.05% Triton X-100/PBS) and once with PBS. Secondary antibodies (dilution 1:200 in 1% normal goat serum/PBS) were added for 1 h and then washed. The samples were then mounted with ProLong Gold antifade reagent with DAPI (Molecular Probes/Invitrogen, CA), following the manufacturer instructions. All incubations and washes were done at 4 or 25°C as required. Confocal microscopy was performed using a Leica SP5 confocal microscope at the Shared Instrumentation facility of department of Hematology at Mount Sinai School of Medicine, NY.