Cd62l bv786

Multiparametric flow cytometry was performed to characterize the generated T cell responses after peptide- or control (no peptides)-stimulation or priming with peptide- or control (no peptide)-pulsed DCs. T cells were washed and resuspended in 100 µL of staining buffer containing 2.5 µg of Human BD Fc Block™ (#130-059-901, Miltenyi Biotec, Bergisch Gladbach, Germany). Cell surface staining of various markers was obtained using the following antibodies: CD3-APC-Cy7 (#560176; clone SK7; BD Biosciences, Franklin Lakes, NJ, USA), CD4-PE-efluor 610 (#61-0049-42; clone RPA-T4; eBioscience, San Diego, CA, USA), CD45RA-APC (#304112; clone HI100; BioLegend, San Diego, CA, USA), CD45RO-BUV395 (#564291, clone UCHL1, BD Biosciences, Franklin Lakes, NJ, USA), CD62L-BV786 (#565312, clone SK11, BD Biosciences, Franklin Lakes, NJ, USA) and CCR7-BV711 (#563712, clone 3D12, BD Biosciences, Franklin Lakes, NJ, USA). Dead cells were gated out using the 7-AAD viability dye (#00-6993-50, eBiocience, San Diego, CA, USA). Data analysis was performed using the BD LSRFortessa X-20 instrument and FlowJo software (BD Biosciences, Franklin Lakes, NJ, USA).

Single cell suspensions from tumors and spleens were incubated for 5 min with F_c-block (BD Biosciences) and then stained with either a myeloid panel of antibodies comprising CD45-BV786 (Clone 30-F11, BD Biosciences), GR1-PE (Clone RB6-8C5, BD Biosciences), CD11b-BV711 (Clone MI/70, BD Biosciences), Ly6C-PerCpCy5.5 (Clone AL-21, BD Biosciences), Ly6G-FITC (Clone IA8, BD Biosciences) and DAPI (Invitrogen) or a lymphoid panel of antibodies comprising CD45-AlexaFlur700 (Clone 30-F11, BD Biosciences), CD3-PE (Clone 145-2C11, eBioscience), NKp46-PE-Cy7 (Clone 29A1.4, eBioscience), CD4-APC (Miltenyi Biotec), CD8-FITC (Miltenyi Biotec), CD44-BV711 (Clone IM7, BD Biosciences), CD62L-BV786 (Clone MEL-14, BD Biosciences), PD-1-BV605 (Clone J43, BD Biosciences) and DAPI (Invitrogen). In some experiments MDCSs were also analyzed for iNOS-PE (Clone CXNFT, eBioscience) expression. Cells were acquired on a BD LSRFortessa and analyzed using FACSDiva.