Phycoerythrin conjugated anti il 17 antibody

At 1 and 3 dpi, single cell suspensions of lymph node cells (LNCs) were prepared from the cervical and axillary lymph nodes of WT and CD69KO mice and stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) plus 2 μg/ml ionomycin for 3 h in the presence of 2 µM monensin (Sigma, St. Louis, MO). Then, the cells were stained with allophycocyanin-conjugated CD4 antibody (Biolegend). After fixing and permeabilizing (Fix/Perm buffer, Biolegend), the cells were further stained with phycoerythrin-conjugated anti-IL-17 antibody and FITC-conjugated anti-IFN-γ antibody (BD Biosciences, San Jose, CA). Analysis was performed with a FACS CantII flow cytometer (BD Biosciences) and FlowJo software (Tree Star Inc., Ashland, OR, USA).

The percentages of CD4⁺ IFN-γ⁺ T cells and CD4⁺ IL-17⁺ T cells in mice cornea and conjunctiva (6 eyes per group) were evaluated using flow cytometric analysis as previously described (21 (link)). Tissues from each group were surgically removed and immersed in PBS. Subsequently, samples were torn apart with scissors and incubated with 0.5 mg/ml collagenase type D (Roche Applied Science) under agitation at 37˚C for 45 min. The samples were disrupted by grinding using a syringe plunger and subsequently passed through a cell strainer with a pore size of 100 µm. Cells were then centrifuged for 7 min at 450 x g at 4˚C. Subsequently, samples were resuspended in PBS containing 1% BSA, then 2 µl of fluorescein-conjugated anti-CD4 antibody (0.5 mg/ml; cat. no. 553651; BD Biosciences), phycoerythrin-conjugated anti-IFN-γ-antibody (0.5 mg/ml; cat. no. 554412; BD Biosciences) and phycoerythrin-conjugated anti-IL-17 antibody (0.5 mg/ml; cat. no. 561020; BD Biosciences) were added for an incubation at 4˚C for 30 min. Phycoerythrin-conjugated rat IgG isotype (BD Biosciences) was used as the control. The percentage of CD4⁺ IFN-γ⁺ and CD4⁺ IL-17⁺ T cells were evaluated using a FACSCalibur cytometer with CellQuest software (version 5.2.1; BD Biosciences).