Ab246535

Cell culture supernatant samples from mcRPE (30DPS) were obtained by incubating volumes of microcarriers containing approximately 0.8 cm² in 48-well plates with 0.5 mL/well of fresh CTS AIM-V medium for 24 h, ±1 h at 37 °C and 5% CO₂. Brightfield whole-well imaging was performed using a Celigo Image Cytometer (Nexcelom Bioscience, Lawrence, MA, USA), and the number of microcarriers per well was counted using the ImageJ Cell Counter plugin (mean ± s.d., 580 ± 132 microcarriers/well). Samples were frozen at −80 °C until the time of processing. Sandwich ELISAs for Pigment Epithelium-Derived Factor (PEDF) and Vascular Endothelial Growth Factor A (VEGF) were performed using commercially available kits according to the manufacturer’s instructions (PEDF: Abcam (Cambridge, UK) #ab246535, VEGF: Abcam #ab222510). Sample dilutions of 80-fold for the PEDF ELISA and 5-fold for the VEGF ELISA were performed to ensure that all results remained in the linear detection range for each assay. Surface area normalization was based on a mean surface area of 0.091 mm² per microcarrier for Cytodex 1 and 0.071 mm² per microcarrier for Cytodex 3 based on diameters of 170 µm and 150 µm, respectively, with spherical surface area $A=4\pi r^2$ [21 ].

The level of serum pigment epithelium-derived factor (PEDF) was measured using commercially available kits according to the manufacturer’s instructions (#ab246535, Abcam, Cambridge, UK). Briefly, 50 µL of assay diluent, human recombinant protein standards, and diluted serum samples were added sequentially to appropriate wells. Subsequently, 50 µL of Antibody Cocktail was added to all wells and incubated at room temperature for 1 h. The wells were then washed three times with washing buffer to remove any unbound detection antibody. Next, 100 µL of TMB Development Solution was added and incubated at room temperature for 10 min. Finally, the reaction was terminated by adding 100 µL of stop solution, and the signal was immediately read at 450 nm.