Tlr4 apc

TLR4 endocytosis was determined as described [25 (link)]. Briefly, primary mouse hepatocytes were treated with SAA1 (2 μg ml⁻¹) or control for 120 min at 37 °C. Cells were subsequently stained with the antibody against TLR4 (TLR4-APC, SA15-21, BioLegend) for 30 min on ice. The cells marked by the antibody were washed three times with PBS and analyzed with FACS Verse (BD Biosciences). The geometric mean fluorescence intensity of TLR4-APC was recorded.

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).