Fluorescein conjugated anti cd4 antibody

Flow cytometry was performed to quantify the number of CD4+C-X-C chemokine receptor type 3 positive (CXCR3+) T cells in the conjunctiva as previously described [31 (link)]. The conjunctival tissues (four eyes per group) were harvested, dipped in phosphate-buffered saline (PBS), teased apart using scissors, and then shaken at 37°C for 60 minutes in the presence of 0.5 mg/mL collagenase type D (Roche Applied Science, Indianapolis, IN, USA). After incubation, the tissues were disrupted by grinding using a syringe plunger and passed through a cell strainer with a pore size of 100 mm. The cells were centrifuged at 1500 rpm for 7 minutes and then resuspended in PBS with 1% bovine serum albumin (BSA). After washing, the samples were incubated with the fluorescein-conjugated anti-CD4 antibody (BD Biosciences, San Jose, CA, USA), phycoerythrin-conjugated anti-CXCR3 antibody (clone 173, BD Biosciences), and isotype control antibody at 37°C for 30 minutes. The phycoerythrin-conjugated rat IgG isotype (BD Biosciences) was used as the control. The number of CD4+CXCR3+ T cells was counted using a fluorescence-activated cell sorting Calibur cytometer with the CellQuest software (both from BD Bioscience, Fullerton, CA, USA).

Flow cytometry was performed for quantification of CD4+CCR5+ T cells from the conjunctiva and cornea with a previously described method [22 (link)]. The tissues were teased and shaken at 37°C for 60 minutes with 0.5 mg/mL collagenase type D. After grinding with a syringe plunder and passage through a cell strainer, cells were obtained, centrifuged, and resuspended in PBS with 1% bovine serum albumin. After washing, the samples were incubated with fluorescein-conjugated anti-CD4 antibody (BD Biosciences, San Jose, CA), phycoerythrin-conjugated anti-CCR5 antibody (BD Biosciences), and isotype control antibody at 37°C for 30 minutes. The number of CD4+CCR5+ T cells was counted by using a FACSCalibur cytomemter with CellQuest software (BD Biosciences).