Live dead cytotoxicity assay

Cell viability was measured using the Live/Dead Cytotoxicity Assay (Molecular Probes). For this assay, both MeOH (shown in Results) and puromycin were to induce death in ARPE-19 cells; similar trends were observed with both. All experiments were done in triplicate and repeated 2 times; similar trends were seen between experiments.

RGCs were isolated from retinas of postnatal day 6 neonatal mice using the Miltenyi Biotec magnetic cell sorting (MACS) system following a published protocol (Huang et al., 2003 (link); Jiao et al., 2005 (link)). Immediately after isolation, cells were incubated with adeno-associated virus (AAV)-GFP or AAV-p58^IPK (Vector Biolabs, Malvern, PA, USA) at 10^∧12 GC/ml following standard procedures approved by the Institutional Biosafety Committee at the University at Buffalo. After 24 h, transduced cells were treated with 1 μg/ml tunicamycin (TM) or a vehicle control (0.05% DMSO) for an additional 16 h. Cell viability was examined using the live/dead cytotoxicity assay (Molecular Probes) following the manufacturer’s protocol. Images were analyzed for live cells (green) and dead cells (red), blind to treatment or genotype (see Supplementary Material).

The fabricated CMMA 3NF scaffolds were seeded with 3T3L1 and MCF7 (3 × 10⁴) in a 24 well plate and cultured in DMEM medium in 5% CO₂ incubator at 37 °C in a humidified environment. The scaffolds were cut into 1.5 cm × 1.5 cm size to fit the cell culture plates and were taken in triplicate and the cell proliferation was analyzed in the alternative days up to 5 days using Dojindo’s cell counting kit-8 (CCK) assay. During 2^nd, 4^th and 6^th day, CCK reagent was added and incubated for 4hrs and absorbances were measured using the microplate reader. Live/Dead cytotoxicity assay (Molecular probes, USA) was done to qualitatively analyze the cytocompatibility of the fabricated 3D scaffolds. The 3T3L1 preadipocytes were grown on the CMMA 3NFs for 6 days and were stained with 200 μl of calcein AM and ethidium homodimer (0.5 μM) for 30 minutes at room temperature. The CMMA 3NFs containing the cells were viewed directly under a fluorescent microscope with a standard fluorescein band pass filter for calcein and a Texas Red® dye filter for ethidium homodimer. The extent of 3T3L1 attachment and proliferation density (day 6) on the CMMA 3NFs were analyzed using the cytoskeletal Actin Green staining (Thermo Fisher Scientific, USA) and Rhodamine B (Molecular probes, USA) the cell nuclei were stained with DAPI (4,6-diamidino-2-phenylindole, Invitrogen, USA, 405 nm, blue).