Phallodin

Polarized ARPE-19 cells grown on transwell filters were maintained in serum free DMEM F-12 Ham for 48 hours. Cells were stimulated with 10% Normal Human Serum or heat inactivated Normal Human Serum (Hi) (56°C 30 minutes) either alone or with human serum albumin (HSA) or CEP-HSA for 24 hours. Where indicated cells were pre-treated for 1 hour with anti-TLR2 blocking antibody (T2.5 Invivogen), IgG control (0.1 µg/ml) or Mal peptide inhibitor (Calbiochem). Supernatants were harvested and assessed for soluble MAC formation by ELISA (Abbexa). Transwell inserts were fixed with 4% Paraformaldehyde for 10 minutes at room temperature, blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature and incubated with anti-mouse-C5b-9 1:25 (Santa cruz) overnight at 4°C. Transwells were washed 3 times in PBS and incubated with goat anti-mouse 647 1:500 (Invitrogen) and Phallodin 1:500 (Invitrogen) for 2 hours at room temperature. Cells were counted stained with Hoechst. Transwells inserts were carefully cut with a sterile blade and mounted on to polysine coated slides (Thermo Scientific) using Mowiol® 4–88. Staining was analyzed using a confocal laser scanning microscope Axio Observer Z1 Inverted Microscope equipped with a Zeiss LSM 700 T-PMT scanning unit and a 40x plan.