Human mfg e8 quantikine elisa kit

The serum concentration of MFGE8 was analysed using sandwich ELISA (Human MFGE8 Quantikine ELISA Kit; R&D Systems, Minneapolis, MN, USA) according to the manufacturers’ protocols. The absorbance was measured at 450 nm on a Microplate Absorbance Spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA), and the data were analysed against a standard curve using Microplate Manager^® Software (Bio-Rad Laboratories).

Passage 3 iPS-derived RPE, fetal RPE, ARPE-19, and RMECs were thawed and seeded at 1.5 × 10⁵ cells per cm² in triplicate onto Matrigel-coated 24-well Transwell® inserts (Corning). Cells were allowed to mature for 30 days and the medium was collected 48 h after the last medium change from the apical to basal compartments. Media samples were then flash frozen in liquid nitrogen and stored at −80 °C. Secreted protein was measured using ELISA as per manufacturer’s recommendations for pigment-epithelial-derived factor (Human PEDF ELISA Kit, BioProductsMD, Middletown, MD, USA, http://www.bioproductsmd.com/) and milk-fat globule-EGF factor 8 (Human MFG-E8 Quantikine ELISA Kit, R&D Systems, Minneapolis, MN, USA, http://rndsystmes.com/). Optical density was measured using a fluorescent plate reader (Synergy H1 Hybrid Multi-Mode Reader).

To validate the mass spectrometry-based protein expression data, Milk fat globule-EGF factor 8 protein (MFGE-8) concentration was measured in the EV-treated cell culture supernatants using a commercially available ELISA kit (Human MFG-E8 Quantikine ELISA Kit, R&D systems) according to the manufacturer’s instructions. Briefly, 150 µL of the cell culture supernatant from each sample was diluted twice with the sample diluent provided with the kit. Then, the assay diluent was added to the precoated 96-well plate, followed by 100 µL of the sample, standard, or control. After two hours of incubation, the wells were washed and incubated with the MFGE-8 conjugate for 2 h. Next, the wells were washed, filled with 200 µL of substrate solution, and incubated for 30 min. Finally, stop solution was added, and the optical density of each well was measured using a microplate reader set to 450 nm (Multiskan FC microplate photometer, Life Technologies, Chongqing, China). The MFGE8 protein concentration changes in the JAr and HEK EV-treated groups from 0 h to 24 h were shown as mean ± SD (fold change expression). Statistical significance was determined using Student’s t-test, and results were considered significant at a p value of <0.05.