Pe conjugated anti cd8a

In vitro cultured CD8⁺ T cells were incubated with 5 μM fluorescent dye Fluo-4 AM (Thermo Fisher Scientific, F14201) for 30 min in PBS at 37 °C. After washing with PBS, cells were labeled with PE-conjugated anti-CD8a (BioLegend, 300908) and Zombie Aqua™ dye (BioLegend, 423101) for 15 min and resuspended in PBS after washing. PMA and CaCl2 were sequentially added to cells while running on an ACEA NovoCyte^TM. Flow data were analyzed with the FlowJo (10.4).

To analyze the population
of T cells within the TME, cells were collected from the tumors on
day 10 following various treatments. This was achieved using a tumor
dissociation kit (Miltenyi Biotec, North Rhine-Westphalia, Germany)
along with a dissociator (GentleMACS, Miltenyi Biotec) to obtain single-cell
suspensions, which were then filtered through a 70 μm strainer
(Smartstrainer, Miltenyi Biotec) to eliminate larger debris. Thereafter,
the resulting single-cell suspensions underwent a blocking step with
antimouse CD16/32 antibodies (101302, 1:25, BioLegend) and were subsequently
stained with the following antibodies: APC/cyanine7- conjugated anti-CD45
(103116, 1:40, BioLegend), FITC-conjugated anti-CD3 (100204, 1:25,
BioLegend), PE-conjugated anti-CD8a (100708, 1:100, BioLegend), and
APC-conjugated anti-CD4 (100412, 1:100, BioLegend) antibodies. The
antibody staining process occurred over a period of 30 min at 4 °C,
followed by 7-AAD viability staining (420404, BioLegend). The stained
cells were then analyzed using a cell sorter (Aurora CS, Cytek Biosciences,
Fremont, CA, USA), and the data were analyzed using FlowJo Software
(Treestar, Ashland, OR, USA).

A total of 2.5 × 10⁶ splenocytes were mixed with purified MARV GP (10 mg/L, from the 293F expression system) were seeded into 12-well plates. After 72 h of culture, cells were collected by centrifugation and resuspended in PBS containing 2% FBS and 0.1% NaN₃. Non-specific Fc receptors were blocked with 1 μg of anti-mouse CD16/CD32 antibody (BioLegend, 101301) per sample. Each sample was stained with 1 μg of APC-Cy7 conjugated anti-CD3e (ThermoFisher Scientific, 47-0031-82), 0.25 μg FITC-conjugated anti-CD4 (ThermoFisher Scientific, 11-0041-82), 0.25 μg PE-conjugated anti-CD8a (BioLegend, 100707), 0.5 μg APC-eFluor780 conjugated anti-B220/CD45R (ThermoFisher Scientific, 47-0452-82), 0.25 μg PE-conjugated anti-CD138 (BioLegend, 142504), 0.5 μg PE-Cy7 conjugated anti-CD80 (BioLegend, 104734), and 0.06 μg APC conjugated anti-CD62L antibody (ThermoFisher Scientific, 17-0621-82) for 40 min on ice. The fluorescence signal was recorded using a flow cytometer (BECKMAN COULTER, CytoFLEX).