Used inFigures 5 and S5
Sender cells and receiver cells were co-cultured on glass-bottom
dishes, in the excess sender configuration, as described above. After 24h of
co-culture, cells were fixed in 4% formaldehyde (diluted in PBS). All
subsequent steps were carried out in blocking solution (2% Bovine Serum
Albumin diluted in PBS). Following 1h of incubation at room temperature,
samples were incubated overnight at 4 C with 1:250 mouse anti-hNotch1
(Biolegend Catalog No. 352014, RRID AB_10899408). Samples were then washed
and incubated in an anti-mouse secondary antibody conjugated to Alexa Fluor
488 (Life Technologies). After room temperature washes, samples were
permeabilized in 0.3% Triton X-100 (Sigma-Aldrich) for 1h. Samples were then
again incubated in 1:250 anti-hNotch1 overnight at 4C, following which they
were incubated in Alexa Fluor 647 conjugated anti-mouse antibody (Life
Technologies).
Sender cells and receiver cells were co-cultured on glass-bottom
dishes, in the excess sender configuration, as described above. After 24h of
co-culture, cells were fixed in 4% formaldehyde (diluted in PBS). All
subsequent steps were carried out in blocking solution (2% Bovine Serum
Albumin diluted in PBS). Following 1h of incubation at room temperature,
samples were incubated overnight at 4 C with 1:250 mouse anti-hNotch1
(Biolegend Catalog No. 352014, RRID AB_10899408). Samples were then washed
and incubated in an anti-mouse secondary antibody conjugated to Alexa Fluor
488 (Life Technologies). After room temperature washes, samples were
permeabilized in 0.3% Triton X-100 (Sigma-Aldrich) for 1h. Samples were then
again incubated in 1:250 anti-hNotch1 overnight at 4C, following which they
were incubated in Alexa Fluor 647 conjugated anti-mouse antibody (Life
Technologies).