αmem without phenol red

The sender and receiver cells were seeded
with a 1:1 ratio in 24-well glass bottom plates (De-Groot). The endogenous
and ff-synNotch cells were seeded 24 hours before imaging, while aa-synNotch
system cells were seeded one hour before imaging. Directly prior to
imaging, the media were replaced with low fluorescence imaging media
(αMEM without phenol red, ribonucleosides, deoxyribonucleosides,
folic acid, biotin, and vitamin B12 (Biological Industries, Israel)
and 100 ng/mL doxycycline (Sigma-Aldrich) was added to the growth
medium to induce ligand expression. For the ff-synNotch system, 250
nM/mL rapamycin (Zotal) was added as well.

A co-culture/monoculture of Fat4-citrine and Ds1-mCherry expressing cells (1.6 × 10⁴ cell/ml) was seeded onto 24-well glass bottom plates (Cellvis, USA) or 35 mm plates (SPL lifesciences, Korea) 12 hr prior the imaging.
For snapshot analysis assay the 24-well plates were covered with 50 ug/ml of Concanavalin A (Sigma Aldrich) to improve cell adherence. Cells were grown for 12 hr and then for induction of Ds1-mCherry, 100 ng/ml doxycycline (Sigma-Aldrich) was added to the growth medium for various periods of time. After that the cells were washed with PBS and fixed for 15 min at room temperature with 2% paraformaldehyde in PBS. To visualize nuclei, the cells were stained with Hoechst Stain solution (Sigma Aldrich) for 5 min.
For FRAP on boundaries and FRAP-TIRF, the cells were seeded on 24-well plates and 35 mm plates, respectively. Directly prior the imaging the media was replaced with low fluorescence imaging media (αMEM without Phenol red, ribonucleosides, deoxyribonucleosides, folic acid, biotin and vitamin B12 (Biological Industries, Israel)). For induction of Ds1-mCherry expression, 100 ng/ml doxycycline (Sigma-Aldrich) was added to the growth medium 12 hr prior to imaging.