Dylight 649

The impact of mycolactone on TLR4 and IFNγR surface expression was measured in primary microglia exposed to increasing doses of mycolactone for 16 h. Microglia were detached with Accutase solution (Sigma) and blocked 20 min with FcR blocking reagent mouse (Mitenyi Biotec, 130-092-575) before being incubated 30 min with either a PE anti-mouse TLR4 (CD284)/MD2 complex antibody (117605, Biolegend) or a PE anti-mouse CD119 antibody (12-1191-82, ThermoFisher) or with the corresponding isotype in PBS 2% SVF at 4°C. SCs exposed to mycolactone for 16 h were detached with Accutase before being labeled with the PE anti-mouse TLR4 (CD284)/MD2 complex antibody. NOS-2 expression was monitored in microglia exposed to increasing doses of mycolactone, 30 min prior to LPS/ IFN-γ stimulation during 16 h (respectively 1 μg/ml and 20 ng/ml). Microglia were fixed (BD Lyse/ Fix buffer, BD Biosciences) and permeabilized (BD Perm/ Wash, BD Biosciences), then incubated for 30 min with anti-NOS2 (Santa Cruz Biotechnology, M-19), then for 20 min with the anti-goat DyLight 649 (Rockland, 605-443-002). Intracellular NOS2 staining was analyzed with the BD Accuri C6 flow cytometer (BD).

An equivalent of one RPE-eyecup of ESMs (1 µg), or one ARPE-19 culture well of ESMs (5 µg) was run on a 4–12% NuPage gel (Invitrogen, ThermoFisher, Waltham, MA, USA), and the electrophoresed proteins were transferred to a nonfluorescent nylon filter. The filter paper was probed with a rabbit anti-FasL antibody (Santa-Cruz Biotechnology, SD, CA, USA), followed by a secondary anti-rabbit-IgG conjugated to DyLight 649 (Rockland, Limerick PA, USA). The stained filters were imaged in a BioRad (Hercules, CA, USA) VersaDoc imaging system using two-color fluorescence to detect RPE65 autofluorescence and immunostained FasL-bands. From the digital images, the band intensities were quantified using Quantity One software (BioRad).

HeLa cells were obtained from the American Type Culture Collection and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Nacalai Tesque, #08458-45) containing 10% fetal bovine serum (Equitech Bio, #268-1). Rabbit polyclonal anti-TOM20 antibody was purchased from Santa Cruz Biotechnology (#sc-11415), and mouse monoclonal anti-GM130 antibody was from BD Biosciences (#610822). Alexa Fluor 555 conjugated anti-mouse IgG (#A31570), Alexa Fluor 555 conjugated anti-rabbit IgG (#A31572), Alexa Fluor 555 conjugated streptavidin (#S21381), Alexa Fluor 647 conjugated streptavidin (#S21374), and Alexa Fluor 647 conjugated wheat germ agglutinin (WGA) (#W32466) were from Thermo Fisher Scientific. Biotinylated WGA (#B-1025) was from Vector Laboratories. DyLight 549 (#S000-42) and DyLight 649 (#S000-43) conjugated streptavidins were from Rockland. HiLyte Fluor 555 (#AS-60666) and HiLyte Fluor 647 (#AS-60667) conjugated streptavidins were from Anaspec. iFluor 555 (#16989) and iFluor 647 (#16996) conjugated streptavidins were from AAT Bioquest. ATTO 647 (#AD647-61) streptavidin was from ATTO-TEC. SPICA dye-conjugated streptavidin was from Fujifilm Wako Chemicals. Biotin-SP AffiniPure goat anti-rabbit IgG (#111-065-144) was purchased from Jackson ImmunoResearch.