Antibody list: Alexa Fluor® 488 anti-human CD41 Antibody (Biolegend, cat# 303723), APC anti-human CD235ab Antibody (Biolegend, cat# 306607), CD66b-Alexa594 (Biolegend, cat# 392907), CD14-BV421 (Biolegend, cat# 325627).
Single hemogenic endothelial cell was seeded in fibronectin coated 96-well plates (Cellvis, cat# P96–1.5P) as described above. Cells were cultured in 100 μL of methylcellulose medium (STEMCELL Technologies, cat# 4435) per well for 8 days before staining for CFU-E and BFU-E, for 14 days before staining for CFU-GM and CFU-GEMM. For staining, antibodies were 1:100 diluted in IMDM (GIBCO, cat# 12440046). 30 μL of diluted antibodies was added to each well on top of the methylcellulose medium (pipette slowly so as not to disturb the hematopoietic colonies). Incubate plates at 37°C overnight before imaging with fluorescence microscope.
Single hemogenic endothelial cell was seeded in fibronectin coated 96-well plates (Cellvis, cat# P96–1.5P) as described above. Cells were cultured in 100 μL of methylcellulose medium (STEMCELL Technologies, cat# 4435) per well for 8 days before staining for CFU-E and BFU-E, for 14 days before staining for CFU-GM and CFU-GEMM. For staining, antibodies were 1:100 diluted in IMDM (GIBCO, cat# 12440046). 30 μL of diluted antibodies was added to each well on top of the methylcellulose medium (pipette slowly so as not to disturb the hematopoietic colonies). Incubate plates at 37°C overnight before imaging with fluorescence microscope.