Pe cy7 anti mouse cd8

The expression of α-gal epitopes was evaluated on SKOV3-gal cells by flow cytometry. Approximately 1 × 10⁶ parental SKOV3 and α1,3GT transfected cells were suspended in 2 % BSA/PBS and incubated with biotinylated bandeiraea simplicifolia isolectin B4 (BS-IB4) lectins, which specifically binds to α-gal epitopes. Then cells were incubated with PE-Cy5 conjugated streptavidin.
The effect of cancer cell vaccine on the immune system of the KO mice was evaluated by analyzing the changes of CD3 + CD4+ and CD3 + CD8+ T cells in spleen. First the spleen was removed, then cut into pieces and grinded gently through a 200 mesh sterile nylon net. The cell suspension was carefully collected and layered on the Ficoll-Paque, PREMIUM (GE Healthcare Life Sciences, USA). The separated splenic mononuclear cells were incubated with fluorochrome-conjugated antibodies directed at the following CD markers: PE anti-mouse CD3, FITC anti-mouse CD4, and PE-Cy7 anti-mouse CD8 (all purchased from eBioscience, San Diego, CA). Gated CD3 positive events were analyzed for CD8+ and CD4+ T cells. Flow cytometry was performed using a FC500 flow cytometer (Beckman Coulter, Fullerton, CA) and analyzed using Beckman Coulter CXP software.

Hearts of mice were minced into 1 mm³ sections and digested with 0.1% collagenase B (Roche Diagnostics GmbH) for 6 min four times in a 37°C water bath (9 (link)). Cell suspensions were obtained by filtering through a cell strainer (40 μm size, BD Falcon) and layered over Ficoll–Hypaque density gradient solution to separate mononuclear cells for flow cytometry. Intracardiac IL-9-producing leukocytes were measured by labeling the harvested cells with the following surface markers: PE anti-mouse CD45, FITC anti-mouse CD4, FITC anti-mouse CD49b, PE-cy7 anti-mouse CD11b, PE-cy7 anti-mouse CD8, or PE anti-mouse Gr-1 antibodies (eBioscience). After washing with PBS, these cells were stimulated with 1 μg/mL ionomycin, 20 ng/mL phorbol myristate acetate (PMA), and 2 μmol/L monensin (eBioscience) for 4 h under 5% CO₂ at 37°C in 24-hole culture plates (Costar). After washing, fixing, and permeabilizing according to the manufacturer’s instructions, the cells were stained with APC anti-mouse IL-9 antibody or isotype control antibody. The stained cells were measured and analyzed by FACScalibur flow cytometry (BD Biosciences).