Used inFigure S5D Recombinant mouse Notch1ext-Fc chimeric protein (R&D
Systems) was used for surface-detection of ligands at a concentration of 10
ug/ml, based on a previously described protocol (LeBon et al., 2014 (link)). Sender cells were first
cultured and induced with 4epiTc for 48h, then transferred from media to
blocking solution (2% FBS in Phosphate Buffered Saline, PBS) for 30 min at
room temperature (RT). Cells were then incubated with recombinant mouse
Notch1ext-Fc protein in binding solution (blocking solution
containing 100 ug/ml CaCl2, R&D Systems) for 45 min at RT.
Following this, cells were washed 3x with binding solution, then incubated
with anti-mouse secondary antibody conjugated to AlexaFluor-488 (1:1000
dilution, Life Technologies) for 30 min. Cells were then trypsinized and
analyzed using flow cytometry.
Systems) was used for surface-detection of ligands at a concentration of 10
ug/ml, based on a previously described protocol (LeBon et al., 2014 (link)). Sender cells were first
cultured and induced with 4epiTc for 48h, then transferred from media to
blocking solution (2% FBS in Phosphate Buffered Saline, PBS) for 30 min at
room temperature (RT). Cells were then incubated with recombinant mouse
Notch1ext-Fc protein in binding solution (blocking solution
containing 100 ug/ml CaCl2, R&D Systems) for 45 min at RT.
Following this, cells were washed 3x with binding solution, then incubated
with anti-mouse secondary antibody conjugated to AlexaFluor-488 (1:1000
dilution, Life Technologies) for 30 min. Cells were then trypsinized and
analyzed using flow cytometry.