Anti cd8 pe cy7

Flow cytometric analysis of B cell populations was performed using our previously described gating strategies8 (link)15 (link)16 (link)17 (link). Following red cell lysis, cells were counted and stained with a cocktail of antibodies for the detection of the indicated populations using anti-CD19-AF700, anti-CD23-PE/Cy7, anti-CD21-E450, anti-CD43-PE/Cy7, anti-GL7-FITC, anti-CD138-PE, anti-B220-E450, anti-B220-APC, anti-IL-10-APC, anti-CD3-FITC, anti-CD4-PerCP and anti-CD8-PE/Cy7 antibodies from eBioscience and Biolegend. Plasma cells were phenotyped as (Dump (CD3, CD4, CD8, CD11b, GR1 & CD11c)⁻ CD138⁺CD19⁻B220⁻) with the Dump⁺ markers identified using biotinylated antibodies and svPE. Alternatively, splenocytes were stimulated with 50 ng/ml PMA (Sigma-Aldrich, UK) plus 500 ng/ml ionomycin (Sigma-Aldrich, UK) and 10 μg/ml LPS (E. coli O111:B4, Sigma-Aldrich, UK) for 1 h before addition of 10 μg/ml Brefeldin A (Sigma-Aldrich, UK) and cells were then incubated for a further 5 h at 37 °C with 5% CO₂. Cells were fixed and washed several times in permeabilization buffer before anti-IL-10 was added in permeabilization buffer and incubated at 4 °C for 30 minutes. Cells were then washed three times with permeabilization buffer and finally with FACs staining buffer before data were acquired using a BD LSR II flow cytometer and analysis undertaken by FlowJo software (TreeStar).

C57BL/6 (WT) mice were purchased from The Jackson Laboratories (Bar Harbor, ME).
Cd244^-/- mice on the C57BL/6 background [12 (link)], were kindly provided by Dr. Raymond Welsh (University of Massachusetts Medical Center, Worcester, MA). Mice were maintained in the Translational Biomedical Research Center of the Medical College of Wisconsin and both genders were used for experiments in an age (6–12 week) and gender-matched manner. anti-CD45R-PE-Texas Red, anti-CD45R-PE, anti-GL7-FITC and anti-CD5-APC were purchased from BD Biosciences (San Jose, CA). CD21-eFluor 450, anti-CD23-PE-Cy7, anti-CD93-biotin, anti-CD4-APC-eFluor 780, anti-CD8-PE-Cy7, anti-TCRβ-FITC, anti-TCRβ-PE, anti-CD11b-Alexa Fluor 488, anti-CD244, anti-Foxp3-PE, anti-CD19-Alexa 700, anti-CD4-PE, anti-IgG-FITC and Streptavidin PE-Cy5.5 were purchased from eBioscience (San Diego, CA). Anti-CD11b-Brilliant Violet 605, anti-CD11c-PE, anti-NK1.1-APC, anti-IgM-APC Cy7, anti-IgD-Pacific Blue, anti-CD38-Alexa Fluor 647, anti-CD138-APC and rat anti-mouse IgG_2a-biotin were purchased from Biolegend (San Diego, CA). Anti-IgM-FITC and the SBA Clonotyping System-B6/C57J-HRP were purchased from Southern Biotech (Birmingham, AL). 4-Hydroxy-3-nitrophenylacetyl (NP)-ficoll, NP-Chicken Gamma Globulin (CGG), NP(24)-PE and NP-BSA were purchased from Biosearch Technologies (Novato, CA).

Freshly harvested splenocytes and cells cultured for 7 days were stained with anti-CD3ε PerCP/Cy5.5 (Clone: 145-2C11, BioLegend), anti-CD-8 PE-Cy7 (Clone: 53-6.7, eBioscience) and anti-GranzymeB AlexaFluor700 (Clone: GB11, BD Biosciences). A MHC class I peptide tetramer specific for influenza virus NP_366-374/D^b conjugated to PE was obtained from the Molecular Biology Core Facility at Trudeau Institute (Saranac Lake, NY). Splenocytes and day 7 effectors were incubated on ice for 60 minutes with the Class I tetramer prior to staining with surface antigens on ice for 30 minutes. Cells were permeabilized according to the BD Cytofix/Cytoperm protocol and subsequently stained for intracellular proteins. Data were acquired on the BD LSR II flow cytometer and analyzed using FlowJo 9.3.3 software.

For the analysis of cell surface markers, single-cell suspensions from the spleens were prepared 1 week after the last dose of DMBA. Cells were permeabilized by 0.5% (w/v) saponin in PBS (containing 0.25% (w/v) bovine serum albumin and 0.02% (w/v) sodium azide). Splenocytes (macrophages, B cells, CD4⁺ T cells and CD8⁺ T cells) were analyzed by staining with an antibody solution containing anti-CD11b-Alexa Fluor 700, anti-B220-Pacific Blue, anti-CD4-Dye647 and anti-CD8-PE/Cy7 (all obtained from eBioscience, Frankfurt, Germany). Tregs were analyzed by staining with anti-CD4-APC/Cy7 and anti-CD25-Dye647 and subsequent incubation with FoxP3 Fixation/Permeabilization working solution (all obtained from eBioscience). Then cells were stained with anti-FoxP3-FITC (eBioscience). All samples were measured using the flow cytometer BD LSR II (BD Biosciences, San Jose, CA, USA).

MDA-MB-436 tumors were collected from huNOG-EXL mice and processed with a Miltenyi MACS Tumor Dissociation Kit on a gentleMACS dissociator (Miltenyi Biotec, Germany), following the manufacturer’s protocol. Single-cell suspensions were labeled with the following antibodies: anti-CD45-eFluor 506 (eBioscience 69–0459–42); anti-CD3-eVolve 655 (eBioscience 86-0037-42); anti-CD4-FITC (eBioscience 11-0049-42); anti-CD8-PE-Cy7 (eBioscience 25-0088-42); anti-FoxP3-eFluor 450 (eBioscience 48-4776-42); anti-Ki67-PerCP-eFluor710 (eBioscience 46-5699-42); and viability dye (eBioscience 65-0865-18). Flow cytometry was performed on an Attune NxT flow cytometer (Thermo Fisher, Waltham, US).