Cd8a pacific blue

Expression of B7-1, ICAM-1, and LFA-3 was determined by flow cytometry. Treated MC38 cells were stained with FITC-labeled antibodies to CD80 (B7-1), CD54 (ICAM-1) and CD48 (LFA-3) (BD Biosciences, San Jose, CA). Cells were incubated with antibodies for 30 min at 4°C. Samples were acquired on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The effect of MVA-TWIST/TRICOM on splenic immune cell populations was examined in tumor bearing and non-tumor bearing BALB/c mice 17 days after receiving two vaccinations with MVA-TWIST/TRICOM or being left untreated (n = 5/group). Vaccination of tumor bearing mice began 4 days post-implantation of 5 × 10⁴ 4T1 mammary tumor cells. Spleens were prepared and stained as described previously [60 (link)], using the following antibodies: CD3e-V500, t-APC, CD8a-Pacific Blue, CD25-FITC, CD44- PerCP-Cy5.5 CD11b-V500, Gr-1-APC, CD11c-PerCP-Cy5.5, CD40-FITC (BD Biosciences); CD62L-PE-Cy7, FoxP3-PE, MHC II-efluor450 (eBioscience, San Diego, CA); and CD49b-PE-Cy7 (Biolegend, San Diego, CA). Tetramer staining (Beckman Coulter, Pasadena, CA) was performed on splenocytes from non-tumor bearing mice following 7 days of in vitro stimulation with 1.0 μg/mL Twist peptide (BALB/c - LYQVLQSDEL). All samples were acquired on a BD Verse flow cytometer. All marker expression was determined using FlowJo software (TreeStar, Inc., Ashland, OR).

To analyze immune cell populations in the presence or absence of cabozantinib, CEA-Tg C57BL/6 mice (n =5/group) were fed control or cabozantinib-containing diet for 10 or 35 days. Spleens were harvested and adjusted to a single-cell suspension. Red blood cells were removed using ACK Lysing Buffer (Quality Biologicals Inc., Gaithersburg, MD). Remaining splenocytes were blocked with mouse Fc Block (BD Biosciences) for 30 min at 4°C, then stained with the following antibodies: CD3e-V500, CD4-AF700, CD8a-Pacific Blue, CD25-FITC, CD11b-V500, Gr-1-AF700, CD49b-FITC, CD19-PE-Cy7, CD11c-PerCP-Cy5.5 (BD Biosciences) and MHC II-APC (eBioscience) for 60 min at 4°C. For intracellular staining of cells with FoxP3-PE, cells were incubated with Fixation/Permeabilization solution for 16 h at 4°C, then incubated with the antibody in Permeabilization Buffer (eBioscience) for 60 min at room temperature. All samples were acquired on an LSR II flow cytometer and analyzed using FlowJo software.