Percp cyanine5.5 conjugated anti mouse cd8a antibody

Tumor-infiltrating T cells were analyzed by flow cytometry. First, tumor tissues were excised on day 27 after tumor re-challenge and digested in collagenase (1 mg/mL in RPMI; Sigma-Aldrich) for 2 h at 37 °C with gentle stirring. Red blood cells were lysed and the resulting tumor cell suspension was passed through a 40-μm strainer. The cells were then stained with the following fluorescent antibodies: APC-conjugated anti-mouse CD3 antibody (BioLegend), PE-conjugated anti-mouse CD4 antibody (BioLegend), and PerCP/cyanine5.5-conjugated anti-mouse CD8a antibody (BioLegend). The percentage of CD3(+)CD4(+) and CD3(+)CD8(+) cells was analyzed using a BD FACSCalibur flow cytometer, and CellQuest Pro and FlowJo software v10.0.7 (BD Bioscience).

The mice shown in Figure 5 were sacrificed on Day 14 after a total of six doses, and the tumor tissues were recovered. Tissues were first cut into small pieces with scissors and washed with PBS. Then, collagenase/hyaluronidase was added to digest the tissues with supplement of DNase I. The digestion proceeded at 37°C with continuous shaking for 30 min before quenching with DMEM supplemented with FBS. The digested suspension then flowed through a cell strainer (100 μm). The recovered cells were washed and resuspended in PBS. Cells were first stained with a Zombie NIR™ Fixable Viability Kit (catalog no. 423106) and blocked with TruStain fcX™ anti‐mouse CD16/32 (catalog no. 101320). Subsequently, the cells were stained with FITC‐conjugated anti‐mouse CD45 antibody (Biolegend, catalog no. 103108) and PerCP/Cyanine5.5‐conjugated anti‐mouse CD8a antibody (Biolegend, catalog no. 100734). The stained cells were washed with PBS and ready for flow cytometry analysis after resuspension in PBS. Single and live cells with surface CD45 and CD8 markers were gated and quantitatively analyzed.

A round-bottom 96-well plate was first prepared by incubation with 100 μl PBS supplemented with 1 μg/mL anti-mouse CD3ε (BioXCell, BE0001-1-5MG) and anti-mouse CD28 (BioXCell, BE0015-1-5MG) the day before CD8⁺ T-cell isolation. The supernatant was discarded before use.
CD8⁺ T cells were isolated from the spleen tissue of adult male C57BL/6 mice with a MojoSort Mouse CD8 T Cell Isolation Kit (BioLegend, 480035) according to the standard protocol. The isolated CD8⁺ T cells were first incubated with reagents from a CFSE Cell Division Tracker Kit (BioLegend, 423801) according to the standard protocol and then resuspended in RPMI 1640 medium (Gibco, 11875093) supplemented with 10% FBS (Biological Industries, 04-001-1A) and 1% Pen Strep (Gibco, 15140122). Then, 20 ng/mL mouse IL2 (Novoprotein, CK24) and IL7 (Novoprotein, CC73) were added, and the concentration of cells was 10⁶/mL.
The isolated CD8⁺ T cells were then incubated in the precoated 96-well plates for 72 hours. After that, the cells were collected and stained with a LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen, L34963), PerCP/Cyanine 5.5-conjugated anti-mouse CD8a antibody (BioLegend, 100733) and PE-conjugated anti-mouse IFN-γ antibody (BioLegend, 163503) as described above. The prepared cell suspensions were analyzed on a CytoFLEX LX from Beckman Coulter.