Pe cy7 conjugated anti cd11c

Epididymal white adipose tissues were chopped with scissors for 4 minutes, followed by addition of 2 mL of collagenase II (SIGMA C6885, 400 U/mL) and incubation at 37 °C for 8 minutes. After digestion, single-cell suspensions were treated with 20 μL of 0.5 M EDTA and filtered using 70 μm meshes. The cells were centrifuged at 385 g for 10 minutes at 4 °C to remove floating adipocytes, and red blood cells were lysed in 1× RBC lysis buffer (eBioscience 00-4300-54) for 2 minutes on ice. After blocking of Fc receptors using TruStain FcX (Biolegend 101320), the cells were stained with the indicated fluorochrome conjugated antibodies. We used a LSRFortessa (BD) or FACSCanto II (BD) system, and analysed flow cytometric data using FlowJo (Tree Star Inc.). The antibodies used for flow cytometry were PE-conjugated anti-CD64 (FcγRI) (Biolegend, 139304), PE-conjugated anti-IgG2a,κ (Biolegend, 400507), PerCP-conjugated anti-CD45 (Biolegend, 103130), PE/cy7-conjugated anti-CD11c (Biolegend, 117318), APC-conjugated anti-CD11b (Biolegend, CD11b), and BV421-conjugated anti-SiglecF (BD Horizon, 562681).

The following mouse-specific IgG monoclonal antibodies were purchased from BioLegend (San Diego): biotin-conjugated anti-MHCII; Brilliant Violet 605-conjugated streptavidin; Pacific Blue–conjugated anti-CD45; allophycocyanin-conjugated anti-F4/80; fluorescein isothiocyanate-conjugated anti-B220; phycoerythrin-conjugated anti-IL-10R and anti-SH; PerCP-Cy5.5-conjugated anti-CD11b; PE-Cy7-conjugated anti-CD11c; and Brilliant Violet 510-conjugated anti-CD103. Fixable Viability Dye eFluor780 was purchased from eBioscience (San Diego). Single-cell suspensions from primary tissues were stained with a suitable combination of fluorochrome-conjugated antibodies and Fixable Viability Dye, fixed in 2% paraformaldehyde in PBS, and examined with a BD LSR II flow cytometer (Becton, Dickinson). The data were analyzed with FlowJo software. For counting of cells by flow cytometry, Flow-Count Fluorospheres (Beckman Coulter, Brea, CA) were used per instructions provided by the manufacturer.

To detect the activation of DCs, mononuclear cells from the spleen or MLNs were stained with PE/Cy7-conjugated anti-CD11c (clone N418), PE-conjugated anti-MHC II (clone M5/114.15.2), APC-conjugated anti-PEDV, FITC-conjugated anti-CD11b (clone M1/70), PerCP-Cy5.5-conjugated anti-CD8α (clone 53-6.7) and PerCP-Cy5.5-conjugated anti-CD103 (clone 2E7). To evaluate memory T-cell responses, mononuclear cells from the spleen or MLNs were stained with FITC-conjugated anti-CD3e (clone 145-2C11), APC-conjugated anti-CD4 (clone GK1.5), PerCP-Cy5.5-conjugated anti-CD8a (clone 53-6.7), PE-Cy7-conjugated anti-CD44 (clone IM7) and PE-conjugated anti-CD62L (clone MEL-14). The PE/Cy7-conjugated anti-CD11c and PerCP-Cy5.5-conjugated anti-CD103 antibodies were purchased from BioLegend (San Diego, CA, USA). The APC-conjugated anti-PEDV antibody was prepared in our laboratory. The PE-conjugated anti-CD62L antibody was purchased from Southern Biotech (Birmingham, AL, USA). The other antibodies were obtained from MultiSciences (Hangzhou, China). All data were collected on a FACSCanto^TM (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo (Version 10.0).