Anti cd11c bv650

Tumor cells isolated from mice were thawed and stained for identification of myeloid or lymphoid cell subpopulations according to the procedure described by Rossowska et al. (30 (link)). Briefly, tumor-derived cells were stained with LIVE/DEAD Fixable Violet Dead Staining Kit (Thermo Fisher) and then stained with cocktails of fluorochrome-conjugated monoclonal antibodies: anti-CD3 PE-CF594, anti-CD19 PE-CF594, anti-CD49b PE-CF594 (all from BD Biosciences), anti-CD45 BV605, anti-CD11b PerCP-Cy5.5, anti-CD11c BV650, anti-F4/80 AlexaFluor 700, anti-Ly6C PE, anti-Ly6G APC-Cy7, anti-MHC II FITC, anti-CD86 PE-Cy7 (all from BioLegend) for myeloid cell identification, and anti-CD45 BV605, anti-CD3 BV650, anti-CD4 FITC, anti-CD8 APC/Fire 750, anti-CD25 PE, anti-CD44 PE-Cy7, anti-CD62L PerCP-Cy5.5 (all from BioLegend) for lymphocytes identification. Then, the cells were fixed using the FoxP3 Fixation Permeabilization Staining Kit (eBioscience). Tumor cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or anti-FoxP3 APC (eBioscience) antibodies, respectively. The analysis was performed using a FACS Fortessa flow cytometer with Diva software (Becton Dickinson).

Primary antibodies were obtained from the following: anti-MADCAM-1 and anti-Zonulen Occludens-1 (ThemoFisher, Rockford, IL, USA), anti-HSC70 (Santa Cruz, Dallas, TX, USA), anti-CD31 (Bioss, Woburn, MA, USA), anti-E-Cadherin (Life Technologies Corp, Carlsbad, CA, USA), and anti-ICAM1 (Cell Signaling, Trask Lane Danvers, MA, USA). Antibodies for flow cytometry were purchased from the following: anti-CD8a-BUV615 (BD Biosciences, Franklin Lakes, NJ, USA), anti-CD11c-BV650, anti-CD103-BV785, and anti-INF-gamma-BUV737 (BioLegend, San Diego, CA, USA), anti-Lymphocyte Peyer’s patch adhesion molecule-1 (LPAM1-BV421 or integrin α4β7; BD Biosciences, Franklin Lakes, NJ, USA), and secondary antibodies Alexa Fluor 488 and 568 IgG (Invitrogen, Eugene, OR, USA). Clindamycin was purchased from Fresenius Kabi (Lake Zurich, IL, USA), and tributyrin, sodium butyrate, and lipopolysaccharide were obtained from Sigma-Aldrich (St. Louis, MO, USA). Primers used for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) were synthesized by Integrated DNA Technologies (Coralville, IA, USA).