Mhc 2 efluor450

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).

Cell suspensions were stained with combinations of following monoclonal fluorescently conjugated antibodies: TCRβ eFluor^® 450 (H57-597, eBioscience, San Diego, CA, USA), CD4 FITC (GK1.5, eBioscience), CD8a PerCP-Cyanine5.5 (53–6.7, eBioscience), CD25 APC (PC61.5, eBioscience), FoxP3 PE (NRRF-30, eBioscience), CD45R/B220 APC-Cyanine7 (RA3-6B2, Biolegend, San Diego, CA, USA), IgD Brilliant violet 650™ (11-26c.2a, Biolegend), CD11b Alexa Fluor^® 700 (M1/70, Biolegend), F4/80 PE (BM8, Biolegend), PD-1 PE-eFluor™ 610 (J43, eBioscience), CTLA-4 PerCP-Cyanine5.5 (UC10-4B9, Biolegend), IL-10 PerCP-Cyanine5.5 (JES5-16E3, Biolegend), LAP PerCP-Cyanine5.5 (TWT-16B4, Biolegend), CD14 FITC (Sa2-8, eBioscience), CD206 APC (MR6F3, eBioscience), MHC-II e Fluor^® 450 (M5/114.15.2, eBioscience). To stain for intracellular murine antigens, cells were first stained for surface antigens, then fixed and permeabilized with intracellular fixation and permeabilization buffer kit (eBioscience) according to the manufacturer’s recommendation. For intracellular IL-10 staining, cells were incubated with PMA (50 ng/ml) and ionomycin (1000 ng/ml) for 2 h, then incubated with Brefeldin A (10.6 μM) and Monensin (2 μM) for 3 h. Data were acquired using BD Fortessa X20 (BD Bioscences, San Diego, CA, USA) and analyzed with FlowJo 7.6 software.