Rat anti mouse cd45 30 f11

γδT cells were isolated from the back skin of Aldara-treated WT or Il12rb2^−/− mice on day 6 post treatment. The whole-cell suspension was stained with the following: rat anti-mouse CD45 (30-F11, BD, 1:800); rat anti-mouse CD11b (M1/70, BioLegend, 1:400); rat anti-mouse CD3 (17A2, eBioscience, 1:100); rat anti-mouse γδ TCR (GL3, eBioscience, 1:400); rat anti-mouse Vγ5 (536, BioLegend, 1:400); rat anti-mouse Vγ4 (UC3-10A6, BioLegend, 1:400) or 17D1 antibodies. Cells were sorted with BD FACS Aria III using 70 μm nozzle. Murine keratinocytes were isolated from naive skin or treated with Aldara for 2 days. The whole-cell suspension was stained with rat anti-mouse CD45 (30-F11, BD, 1:800), rat anti-mouse CD34 (RAM34, eBioscience, 1:100) and rat anti-mouse/-human CD49f (GoH3, BioLegend, 1:300) antibodies. Cells were sorted with BD FACS Aria III using 100 μm nozzle.

A mouse LP dissociation kit (Miltenyi Biotec) was used to isolate cells from the small intestine according to the manufacturer's protocol, with a modification at the final dissociation step where cells were gently crushed through a 70 μm cell strainer immediately following enzymatic digestion to generate a single cell suspension. Cells were centrifuged for 10 min at 1,500 rpm and the cell pellet was resuspended in PBS containing 0.5% BSA. Cells were counted using a hemocytometer prior to staining. Cells were stained with fixable viability dye eFluor506 (ThermoFisher) and Fc receptors were blocked with anti-mouse CD16/32 (2.4G2; BD Biosciences). Surface staining included rat anti-mouse CD45 (30-F11; BD Biosciences), rat anti-mouse B220 (RA3-6B2; BD Biosciences) and rat anti-mouse Siglec F (E50-2440; BD Biosciences). Cells were fixed and permeabilized using BD CytoFix/CytoPerm (BD Biosciences) and incubated with rat anti-mouse IgA (C10-1; BD Biosciences). Liquid counting beads (BD Biosciences) were added to samples prior to acquiring data on a CytoFLEX flow cytometer (Beckman Coulter) to enable total cell counts of cells falling within IgA⁺B220⁻CD45⁺ live gates according to manufacturer's protocol. Data were analyzed using CytExpert software (Beckman Coulter).