Pe conjugated anti mouse ly6g

The following antibodies were purchased from eBioscience: Pacific Blue conjugated anti-mouse CD4 (clone: RM4-5), FITC conjugated anti-mouse CD45.1 (clone: A20), biotin conjugated anti-mouse CD3 (clone: 17A2), FITC conjugated anti-mouse CD3 (clone: 145-2C11), APC conjugated anti-mouse CD317 (BST2, PDCA1) (clone: eBio927), Pacific Blue conjugated anti-mouse CD11c (clone: N418), FITC conjugated anti-mouse MHCII (I-A) (clone: NIMR-4), biotin conjugated CD317 (BST2, PDCA1) (clone: eBio927), PerCP-Cy5.5 conjugated anti-mouse Ly-6G (Gr-1) (clone: RB6-8C5), streptavidin conjugated PerCP-Cy5.5. The following antibodies were from BD Pharmingen: PE-Cy7 conjugated anti-mouse CD45 (clone: 30-F11), PerCP conjugated anti-mouse CD4 (clone: RM4-5), PE conjugated anti-mouse CD19 (clone: 1D3), APC-Cy7 conjugated anti-mouse CD11b (clone: M1/70), Biotin conjugated anti-mouse Ly-6C (clone: AL-21), PerCP-Cy5.5 conjugated anti-mouse CD45R (B220) (clone: RA3-6B2), PE conjugated anti-mouse Ly-6G (clone: 1A8), PerCP-Cy5.5 conjugated Ly-6C (clone: AL-21), streptavidin conjugated V500. Diphtheria toxin (DT, isolated from Corynebacterium diphtheriae) was purchased from Sigma-Aldrich (St Louis, MO). Mouse Th1/Th2/Th17 Cytokine kit (BD Biosciences, California, USA) was obtained from BD^TM Cytometric Bead Array (CBA).

Mice were sacrificed and lung tissues obtained were minced using scissors and digested with a mixture of collagen‐Ⅰ and collagen‐IV. The suspended cells were filtered through a 70 μm nylon mesh, centrifuged at 1500 rpm for 5 min, and washed thrice with Phoshhoric acid buffer (PBS). Blood cells of the mice were directly isolated and washed three times with PBS. The cells for flow cytometry were stained with an antibody for 30 min at 4°C. Macrophages were stained with the following antibodies: Pacific Blue anti‐mouse CD11b (1:100; BD Biosciences), PE‐conjugated anti‐mouse CD206 (1:100; BD Biosciences), Allophycocyanin (APC)‐conjugated anti‐mouse F4/80 (1:100; BD Biosciences), PerCP‐CY5.5‐conjugated anti‐mouse CD45 (1:100; BD Biosciences) and Fluorescein isothiocyanate (FITC)‐conjugated anti‐mouse CD11c (1:100; BD Biosciences). Monocytes in blood were stained with the following antibodies: PE‐conjugated anti‐mouse Ly6G (1:100; BD Biosciences), APC‐conjugated anti‐mouse CD11b (1:100; BD Biosciences), PerCP‐CY5.5‐conjugated anti‐mouse CD45 (1:100; BD Biosciences) and FITC‐conjugated anti‐mouse Ly6G (1:100; BD Biosciences). The cells were analysed by flow cytometry using FlowJo software (Tree Star, Inc.).

Retinas were dissected from freshly collected eyes and digested with Liberase^TM (Sigma‐Aldrich, CAT#5401127001, 0.26 U/ml) and deoxyribonuclease I (Sigma‐Aldrich, CAT#D4527, 10 mg/ml) in PBS (1 ml per one retina) at PBS at 37°C for 30 min. The reaction was stopped by adding SVF (final 1%) and centrifuged at 400 × g at 4°C for 10 min. The pellet was further washed once with FACS buffer (0.5% BSA/0.5 mM EDTA in PBS) and stained with APC‐conjugated anti‐mouse CD45 (1:300, BioLegend, CAT#103112), BV510‐conjugated anti‐mouse CD11b (1:300, BioLegend, #CAT101263), and PE‐conjugated anti‐mouse Ly6G (1:300, BD Pharmingen, #CAT5551461) antibodies for 1 h on ice. The retinal cells were washed twice and treated with DAPI to exclude dead cells. CD31⁺/CD45⁻/TER119⁻ cells were analyzed using BD FACSAria™ II (BD Biosciences).