Caspase 3

Some paraffin sections obtained from implant regions were processed for immunocytochemistry against pan-cytokeratin (1:100; catalog number ab11213, Abcam), ezrin (1:50; CST), MCT-1 (1:50; LifeSpan Biosciences), Ki67 (1:50; Sigma), phosphohistone H3 (1:50; Millipore), caspase-3 (1:50, Promega), and human-specific antibody SC121 (1:50; Stem Cells) to verify the presence of human RPE cells on the subretinally implanted cell carriers. Pan-cytokeratin was visualized with the Biotin-ExtrAvidin-AEC chromogen system. All other primary antibodies were visualized using Alexa Fluor secondary antibodies as mentioned above.

Commercial Caspase Activity Assay kits were used to assess the activities of caspase‐3 (Promega, Madison, WI, USA) and caspase‐8 and caspase‐9 (Beyotime, Shanghai, China). An equal amount of total protein extract was incubated at 37°C overnight with either Ac‐DETD‐PNA for the caspase‐3 assay, Ac‐IETD‐pNA for the caspase‐8 assay or Ac‐LEHD‐pNA for the caspase‐9 assay. Release of pNA was determined by absorbance at 405 nm. The relative activity of caspases was calculated as follows: caspase activity = (mean experimental absorbance/mean control absorbance) × 100%. All procedures were performed three times.
Apoptosis was detected by fluorescein isothiocyanate (FITC)‐Annexin V/propidium iodide (PI) double staining (Invitrogen). After 48 h of treatment with SP or aprepitant, the cells were washed twice with PBS. The cells were labelled with FITC‐Annexin V and PI for 10 minutes in the dark and cellular fluorescence was measured using a FACScan flow cytometer (Becton Dickinson). Each experiment was performed in triplicate.31