Anti cd62l percp cy5

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).

Cells were stained with the following antibodies (all from eBioscience, except where stated); anti-CD4-BV650 (BioLegend, San Diego, CA, USA), anti-Foxp3-(ef450/FITC), anti-CD11b-AF700 (Biolegend), anti-αv-PE, anti-CD44-APC-Cy7, anti-CD62L-PerCP-Cy5.5, anti-Ki-67-PE-Cy7, anti-KLRG1-ef450, anti-CD45.1-APC, anti-CD51-PE, anti-GM-CSF-PE, anti-IFN-γ-APC, anti-IL-17-PerCPCy5.5. Rat IgG2a and rat IgG1, conjugated to respective fluorophores, were used as isotype control antibodies. For intracellular cytokine staining, cells were re-stimulated ex vivo with 20 µg/ml pOVA overnight, and Brefeldin A added for the last 4 h of incubation. Samples were stained with a fixable viability marker (conjugated with eFluor455, eBioscience) prior to surface staining. For subsequent intracellular antigen staining, samples were washed once in FACS buffer (PBS, 2% FCS, 0.01% NaN₃) and then processed according to manufacturer’s instructions (eBioscience for transcription factors, BD for intracellular cytokines). Flow cytometric data were acquired using a BD LSR Fortessa cell analyzer (BD) and data analyzed using FlowJo software (Treestar version 3.2.1, Ashland, OR, USA).