Fitc labeled cd8

Blood was collected in heparinized tubes from the retro-orbital plexus to assess CD4, CD8, CD14, and TLR-4 surface markers. FITC- or PE-labeled CD4, PE- or FITC-labeled CD8, APC-labeled TLR-4, and PE/Dazzle-labeled CD14 (BioLegend, USA) monoclonal antibodies were used to determine the percentage of CD4⁺, CD8⁺, TLR-4⁺, and CD14⁺cell surface receptors, respectively. These were added to 100 µL of blood lysed with a lysing buffer (BioLegend, USA). Approximately 20 µL of the fluorescently labeled monoclonal antibodies were added to the suspension of blood leucocytes (CD4 FITC, CD8 PE, CD14 PE/Dazzle-labeled, and TLR-4 APC, BioLegend, USA). These were then incubated at room temperature for 30 min. To stain the intracellular markers IL-17A, NF-κB p65, and TNF-α, the cells were permeabilized, subjected to fixation by standard buffers (BioLegend, USA), and stained with intracellular APC-IL-17A, FITC-NF-κB p65, and PE-TNF-α antibodies (BioLegend, USA; Santa Cruz, Dallas, USA), (Ahmad, Attia, Zoheir, Ashour & Bakheet, 2014 (link)). After centrifugation at 300g for 10 min, the samples were kept at 4 °C until flow cytometry analysis was conducted. Immunostained leukocytes in each sample were acquired using an FC 500 flow cytometer; 10,000 events/sample were then analyzed using CXP software (Beckman Coulter, USA).

Fresh tumor digests (FTD) were prepared using the gentleMACS tumor dissociator (Miltenyi Biotec, Auburn, California, USA), according to the manufacturer’s instructions. Cell counting was performed by manually using a hemocytometer. FTD (2–5×10⁶ cells/tube) were cryopreserved with CP-1 (Kyokutoseiyaku, Tokyo, Japan) and kept in a deep freezer (−135°C) until use. Cryopreserved FTD (2–5×106 cells) were thawed in RPMI and then stained after blocking Fc receptors using Human BD Fc Block (BD Biosciences, San Jose, California, USA). The following monoclonal antibodies (mAbs) were used: BV421-labeled CD3, APC-labeled CD4, FITC-labeled CD8, BV711-labeled CD103, PE-labeled CD39, and BV786-labeled PD-1 (all from BioLegend, San Diego, California, USA). SYTOX AADvanced Dead Cell Stain Kit (Thermo Fisher, Waltham, Massachusetts, USA) or Zombie NIR Fixable Viability Kit (BioLegend) was used to exclude dead cells. Stained cells were analyzed using an LSRFortessa X-20 flow cytometer (BD Biosciences) and data processed using FlowJo V.10.0.7 (BD Biosciences).