Ab46074

CS/CS-CNC hydrogels were prepared as previously described with a cell loading density of 10⁶–10⁷ iNSC-s-TRAIL/mL of hydrogel (100 μL/mold, n = 7) and incubated at 37 $°$ C/5%CO₂. Hydrogel samples were prepared in individual molds and subsequently seeded in a 12-well plate and supplemented with standard cell culture media for 24 h. Following incubation, 1 mL aliquot of TRAIL conditioned media was collected. To measure the amount of TRAIL released from the cell-laden hydrogel scaffolds, an in vitro Human TRAIL Enzyme-Linked Immunosorbent Assay (ELISA) was performed following manufacturer’s protocol (Abcam ab46074, Cambridge, UK). The absorbance from each well was quantified using a microplate reader and the concentration of secreted TRAIL (s-TRAIL) in each sample was determined based on an established standard curve.

Immune cell chemotaxis assays were performed using 1 × 10⁶ autologous PBMCs placed in 5 μM pore size transwell inserts. Assays were performed for 24 h at 37°C and 5% CO₂. Migrated cells present in the lower chamber were removed by pipetting and characterized by flow cytometry. Zombie Aqua viability stain (BioLegend 423101) and antibodies directed toward CD19 (BioLegend, 302218), CD3 (BioLegend, 300424), CD56 (Becton Dickinson, 335791), and CD14 (Becton Dickinson, 563372) were used to identify immune cell populations. All samples examined by flow cytometry have been treated with Fc block (BD, 564219) prior to staining. ELISAs for CCL2 (R&D Systems, DY279), CCL3 (R&D Systems, DY270), CCL4 (R&D Systems, DY271), CXCL10 (BioLegend, 439904), and TRAIL (Abcam, ab46074) were performed according to the manufacturers' protocols.