Cd3 apc 17a2

BM was collected by flushing femurs and tibiae using phosphate-buffered saline (PBS) 1X (no calcium/no magnesium). BM cells were stained for flow cytometry analyses using the following antibodies: CD45.1 PE-Cy7 (A20, eBioScience), TER119 PE-Cy5 (TER-119, BD Biosciences, Franklin Lakes, NJ, USA), CD11b PE (M1/70, Biolegend), Ly6C BV605 (AL-21, Fisher Scientific), CD3 APC (17A2, eBioScience), CD19-PB (6D5, Biolegend), and eFluor 780 viability Dye (eBioScience). Stained cells were acquired using Cytoflex (Beckman Coulter) and analyzed using FlowJo software (FlowJo).

Eyes were harvested, and corneas were isolated under a dissecting microscope at various times postinfection. Corneas were digested in 400 U/ml collagenase II (Life Technologies) for 1.5 h at 37°C, homogenized with an Eppendorf micropestle, triturated with a pipettor, and filtered through a 70-µm mesh filter before flow cytometry staining. Cervical lymph nodes were dissected, similarly homogenized, and filtered before flow cytometry staining. Blood was harvested by submandibular bleed, and erythrocytes (RBCs) were lysed using Gey’s solution. The following antibodies were used for flow cytometric analysis: Ly6G-fluorescein isothiocyanate (FITC) (1A6), CD4-FITC (GK1.5), CD69-phycoerythrin (PE) (H1.2F3), CD8α-PE (53-6.7), CD11c-FITC (HL3), and GR1-peridinin chlorophyll protein (PerCP)/Cy5.5 (RB6-8C5), all purchased from BD Biosciences (San Jose, CA). Ly6C-PerCP/Cy5.5 (HK1.4), CD11b-allophycocyanin (APC) (M1/70), CD3-PerCP/Cy5.5 (17A2), CD45-APC (30-F11), and CD19-PE (6D5) were purchased from BioLegend (San Diego, CA), and CD3-APC (17A2) was purchased from eBioscience (San Diego, CA). Samples were analyzed using the BD Accuri C6 flow cytometer, and the data were analyzed using CFlow software (Accuri).