Pe cy7 anti mouse ly 6c antibody

Ubiquitin-Cre^ERt2; tdTomato^LSL mice were treated with tamoxifen (1 mg IP Q48H, three injections total). Two weeks after the first tamoxifen injection, mice were sacrificed and bone marrow cells were isolated by flushing with ice cold PBS using a 25G needle. 10⁶ bone marrow cells were protected from light and stained on ice for 30 min with the following primary antibodies in a 10 µL staining volume. APC anti-mouse/human CD 11b antibody (Biolegend, San Diego, CA), FITC anti-mouse CD3 antibody (Biolegend, San Diego, CA), and PE/Cy7 anti-mouse Ly-6C antibody (Biolegend, San Diego, CA). After staining, cells were washed twice with FACS buffer (PBS plus 2% heat inactivated fetal bovine serum) and analyzed on a SORP 8 Laser BD LSR flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ).

The BAL cells were pelleted by centrifugation at 400 g for 5 minutes, then RBCs were lysed. The cells were resuspended in PBS and cell counts were quantified using an automated cell counter (DeNovix CellDrop^™). For flow cytometry, the cells were pelleted in v-shaped-bottom 96-well plates. The panel of BioLegend antibodies used for flow analysis was as follows, used at 1: 400: FITC anti-mouse MERTK (Mer) antibody (clone: 2B10C42; BioLegend); Alexa Fluor^® 700 anti-mouse major histocompatibility complex class II (MHCII; I-A/I-E) antibody (clone: M5/114.15.2; BioLegend); allophycocyanin (APC)/cyanine 7 (Cy7) anti-mouse CD11b antibody (clone: M1/70, BioLegend); Brilliant Violet 510^™ anti-mouse Ly-6G antibody (clone 1A8; BioLegend); Zombie Aqua^™ Fixable Viability Kit, Brilliant Violet 605^™ anti-mouse CD11c antibody (clone: N418; BioLegend); Brilliant Violet 650^™ anti-mouse F4/80 antibody (clone: BM8; Biolegend); phycoerythrin (PE)-CF594 rat anti-mouse Siglec-F (clone: E50–2440; BD Biosciences); and PE/Cy7 anti-mouse Ly-6C antibody (clone: Hk1.4; BioLegend). The samples were acquired using the NovoCyte Quanteon flow cytometer (Agilent Technologies) and flow data were analyzed using FlowJo^™ version 10.

AT-infiltrating stromal cells were stained with FITC anti-mouse CD3ε antibody, PE anti-mouse CD4 antibody, PE/Cy7 anti-mouse CD25 antibody, APC anti-mouse CD8a antibody, FITC anti-mouse CD127 antibody, PerCP/Cy5.5 anti-mouse CD4 antibody, APC anti-mouse CD25 antibody (these antibodies were purchased from Biolegend, San Diego, CA, USA), PE anti-mouse/Foxp3 antibody (Invitrogen, Carlsbad, CA, USA), PE anti-mouse CD11b antibody (Biolegend), PE/Cy7 anti-mouse Ly-6C antibody (Biolegend, San Diego, CA, USA) and APC anti-toll-like receptor 4 (TLR4) antibody (Invitrogen, Waltham, MA, USA), and analyzed using an Accuri C6 (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).