HeLa cells were stored at 37°C and 5% CO in T25 flasks in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 1% antibiotic (25units/mL penicillin, 25µg/mL streptomycin)(Invitrogen). For alamarBlue and Live/Dead assays, 96-well glass bottom dishes (Cellvis, Mountain View, CA) were salinated as previously described and 50µL collagen hydrogels were cast in the bottom of the wells and functionalized with EL222-SNAP. Cells were plated at a density of 10,000 cells/well in and monitored over 7 days. Metabolic activity was visualized using media supplemented with 10% alamarBlue solution following manufacturers protocol. 100uL samples of media were taken after a 1 hour incubation at 37°C and fluorescence signal (Ex/Em 560nm/590nm) was read with a plate reader (Biotek). After incubation in alamarBlue-supplemented media, cells were washed with PBS, DMEM, and replenished with fresh media.
Live versus dead cells were quantified using a LIVE/DEAD Viability/Cytotoxicity Kit (Thermo). Cells were washed once with PBS and incubated in 2µM Calcein-AM and 4 µM ethidium homodimer-1 for 45 minutes at 37°C prior to imaging. Images were captured on a Nikon Super Resolution Spinning Disk Confocal microscope and live/dead cells were identified by custom FIJI macros or by hand, depending on cell density.
Live versus dead cells were quantified using a LIVE/DEAD Viability/Cytotoxicity Kit (Thermo). Cells were washed once with PBS and incubated in 2µM Calcein-AM and 4 µM ethidium homodimer-1 for 45 minutes at 37°C prior to imaging. Images were captured on a Nikon Super Resolution Spinning Disk Confocal microscope and live/dead cells were identified by custom FIJI macros or by hand, depending on cell density.