Pe labeled cd4

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).

The cells were stimulated with eBioscience Cell Stimulation Cocktail (2 μg/mL) (eBioscience) for 5 h, collected, centrifuged, resuspended in FACS buffer, stained with PE-labeled CD4 (Biolegend), and incubated at 4 °C in the dark for 20 min. In 1 × Cytofix/Cytoperm Buffer (BD) suspension cells, 4 °C avoid light incubation for 30 min, 1 × Permeabilization Wash Buffer (BD) fixed and 500×g centrifugal 5 min. 1 × Permeabilization Wash Buffer after heavy rupture in suspension cells, according to the instruction manual to join APC IL-17A (Biolegend) dyeing was carried out on the intracellular cytokines, 4 °C avoid light incubation for 30 min, flow cytometry was used for detection.