Fluorochrome conjugated monoclonal antibodies

Cell surface staining was performed in ice-cold PBS containing 2% FBS and 2 mM EDTA (cytometry buffer) using appropriate fluorochrome-conjugated monoclonal antibodies and corresponding isotypes, all purchased from BD Biosciences (Table 3). A total of 1.5 to 2×10⁵ cells per experimental condition were resuspended in cytometry buffer containing 2 µg/mL of FcBlock (anti CD16/CD32) for 20 min at 4°C, then stained with 1 µg/mL of antibody or control isotypes for 30 min on ice in the dark. Next, cells were washed with cytometry buffer, and fixed in BD cell fix buffer (BD Biosciences) or in 4% paraformaldehyde solution before acquisition through a BD FACSCanto™ II Cell Analyzer (IPNC) or a CytoFLEX flow cytometer (Beckman Coulter). For cell viability analysis, cells were incubated with eBioscience™ Fixable Viability Dye eFluor 780 for 5 min on ice, washed in cytometry buffer before fixation. Positive eFluor dead cells were excluded from the analysis. At least 30,000 events were acquired for the phenotyping. Compensations were performed using UltraComp eBeads (Thermofisher Scientific) stained positively with each of the antibodies used in the experiment. Baseline photomultiplier tube voltages of the instrument were set using unstained cells, showing the background fluorescence of the experimental samples. Data were analyzed using FlowJo v10 analysis software package.

Cells were incubated with 24G2 cell supernatant for 20 minutes at 4°C before incubation with fluorochrome-conjugated monoclonal antibodies (BD Pharmingen, Oxford, UK) against cell surface markers including, CD4 (553051, 1/100), CD8 (553029, 1/100), CD11b (557657, 1/400), Ly6G (551461, 1/100), Ly6C (553104, 1/100) and CD45 (552848, 1/1000) at 4°C for 20 minutes. A streptavidin secondary antibody was added to all samples, (48-4317-82, eBioscience, San Diego, USA, 1/1000) then cell suspensions were acquired using a 3-laser BD LSR-II flow cytometer (BD Cytometry Systems, Oxford, UK). Analysis was performed using FlowJo software (Treestar, San Carlos, California, USA). Cell numbers were calculated by reference to a known cell–standard, as previously reported [14 (link)]. In brief, splenocytes at a series of known cell concentrations were acquired using a fixed and stable flow rate for 45 seconds. Based on total cell number acquired during this time, a standard curve was generated and used to interpolate cell concentrations of ocular infiltrating cells acquired at the same flow rate and time.

HMEC spheres were grown in various media as previously described for cell proliferation assay [78 (link)]. Harvested spheres were trypsinized and cells were diluted to 10⁶ cells/ml. Combinations of fluorochrome-conjugated monoclonal antibodies obtained from BD Biosciences (San Diego, CA, USA) against human CD44 (FITC) and CD24 (Alexa 647) or their respective isotype controls were added to the cell suspension at concentrations recommended by the manufacturer and incubated at 4 °C in the dark for 30 to 40 min. The labeled cells were washed in the wash buffer and DAPI was added to gate the live cells during the flow cytometry analysis using LSRII (BD Biosciences). Results were analyzed using ModFit Lt V3 software (Verity Software House).