Fitc conjugated anti ly6g clone 1a8

PMN and lymphocyte cell populations within lacrimal glands, draining lymph nodes, and corneas were assessed by flow cytometry as previously described ¹⁴ . Anti-FcR mAB (BD PharMingen, San Diego, CA) was used to block Fc receptors for 10 minutes before samples were incubated for 30 minutes in titrated amounts of fluorescent-labeled antibodies: FITC-conjugated anti-Ly6g (1A8 clone; BD PharMingen, San Diego, CA, USA) for PMN; PE-conjugated anti-CD45 (MEC 13.3 clone; BD PharMingen) for leukocytes; PE-conjugated anti-CD3 (500A2 clone; BD PharMingen) for T cells; FITC-conjugated anti-CD4 (RM4–5 clone; BD PharMingen) for activated CD4⁺ T cells; APC-conjugated anti-IFN-γ (XMG1.2; TONBO biosciences); FITC-or APC conjugated anti-IL17 (eBio17B7; ebioscience); APC conjugated anti-Foxp3 (3G3; TONBO biosciences). Live cells were sorted using a high-speed sorter (MoFlo, SX DakoCytomation, Inc., Fort Collins, CO, USA).

Lacrimal glands were digested with 2mg/mL collagenase D (Roche Diagnostic Corp.) and 0.5 mg/mL DNase I (Roche Diagnostic Corp.) in FCS-containing RPMI for 1 h at 37°C. Single-cell suspensions from the digested samples and draining lymph nodes were prepared with a 40-μm cell strainer (BD Falcon; Becton-Dickinson, Franklin Lakes, NJ). Fc receptors were blocked with anti-FcR mAb (BD PharMingen, San Diego, CA) and cells were then incubated with titrated amounts of fluorescent-labeled antibodies: FITC- conjugated anti-Ly6g (1A8 clone; BD PharMingen, San Diego, CA, USA) for PMN; PE-conjugated anti-CD45 (MEC 13.3 clone; BD PharMingen) for leukocytes; PE-conjugated anti-CD3 (500A2 clone; BD PharMingen) for T cells; FITC- conjugated anti-CD4 (RM4-5 clone; BD PharMingen) for activated CD4⁺ T cells; APC- conjugated anti-IFN-γ (XMG1.2; TONBO biosciences); FITC- or APC conjugated anti-IL17 (eBio17B7; ebioscience); APC- conjugated anti-Foxp3 (3G3; TONBO biosciences). Isotype control was stained with the appropriately matched antibodies. Data were analyzed with FlowJo (TreeStar, Ashland, OR, USA) software. The percentage of stained cells in the samples was calculated with respect to isotype control staining. Cell sorting was performed using a high-speed cell sorter (MoFlo, SX DakoCytomation, Inc., Fort Collins, CO, USA). Each flow cytometry experiment was performed at least three times.