Anti cd90

Antibody staining was performed on tissue single-cell suspensions for 20-30 minutes at 4°C in Hanks' Balanced Salt Solution staining buffer and then washed twice with cold PBS. The sole exception was CD127, where staining was performed at room temperature for an hour. To measure cytokine production, cells were stimulated in vitro with Phorbol 12-myristate 13-acetate (PMA), ionomycin (Iono), and Brefeldin A (BFA) for 2 hours and 30 minutes. For intracellular staining, cells were fixed for 30 minutes with the eBioscience Fixation/Permeabilization kit as described by the manufacturer and stained intracellularly for at least 1 hour with anti-CD4, anti-CD8, anti-CD90.2, anti-TCRβ, anti-IFN-γ anti-IL-17A and anti-IL17F. Antibodies for flow cytometry were purchased from BD Biosciences, BioLegend, or eBiosciences. Antibodies used were conjugated to FITC, AF488, PE, PerCP-Cy5.5, PCP-eFluor 710, PeCy7, Alexa Fluor 780, Pacific Blue, BV605, BV650, BV 510, BV785, eFluor 450, APC, Alexa Flour 647, PE Texas Red, PE-CF594. DAPI or Live/Dead fixable stain (Life Technologies) was used to exclude dead cells in all experiments. Flow cytometric data was acquired on an LSR II or LSR Fortessa (BD Biosciences) and analyzed using the FlowJo software (Tree Star, Version 9).

Frozen sections (10 μm) were prepared from tissue embedded in optimal cutting medium (OCT 4583, Sakura Finetek, Torrance, CA). Sections were fixed in 4% paraformaldehyde, blocked with horse serum containing 2.5% fraction V BSA for 1h, and then stained with anti-F4/80 (AbD Serotec, Oxford, UK) or anti-CD90.2 (eBiosciences, San Diego, CA) antibodies at 4°C overnight. After three washes with PBS, sections were stained with Alexa fluor 594-conjugated antibodies (Invitrogen, Grand Island, NY) for 20 min. Nuclei were stained with Hoechst for 5 min. Sections were examined with TCS-SP2 AOBS Confocal Laser Scanning Microscope (Leica).

We used the methods detailed in Tran et al. (2019) (link) for dissociation and FACS sorting of RGCs. Briefly, retinas were dissected in AMES solution (equilibrated with 95% O2/5% CO2), digested in papain, and dissociated to single cell suspensions using manual trituration in ovomucoid solution. For a concentration of 10 million cells per 100μl, 0.5μl of 2μg/μl anti-CD90 (conjugated to various fluorophores) (Thermo Fisher Scientific) was used to stain (15 minutes incubation), washed with an excess of media, spun down and resuspended again in AMES+4%BSA to a concentration of ~7 million cells per 1 ml. Just prior to FACS the live cell marker calcein blue was added. RGCs were collected based on high CD90, GFP and, in some cases, micro-Ruby co-expression. For 10X experiments, cells were collected into ~100ul of AMES+4%BSA per 25,000 sorted cells. Following collection cells were spun down and resuspended in PBS+0.1% non-acetylated BSA at a concentration range of 500–2000 cells/ul for droplet-based scRNAseq per manufacturer’s instructions (10x Chromium). For SS2 experiments, single cells were collected into 96 well plates filled with 5μl of TCL lysis buffer, containing 1% BME, spun down and frozen at −80°C till further processing.