Anti tlr4 pe

Peripheral blood mononuclear cells, containing 10⁶ monocytes/mL, were distributed (500 μL) into polystyrene tubes (BD Labware) followed by incubation with culture medium alone or culture medium plus 50 μg ATF for 6 h at 37 °C under 5% CO₂. Cells were washed and labeled with anti-CD14 PerCP-Cy^TM5.5 mAb (BD-Pharmingen) and mAbs for each receptor: anti-MR-FITC (BioLegend), anti-TLR2-FITC (BioLegend), and anti-TLR4-PE (BioLegend), according to the instructions of the manufacturers. Expression of β-glucans receptor (βGR) was evaluated by incubation with the primary mAb – GE2 – generously provided by Dr. Gordon Brown, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa, that recognizes both βGRA and βGRB isoforms of this receptor followed by incubation with a FITC-labeled secondary antibody (Biolegend) [26 (link)]. After incubation for 15 min. at room temperature, cells were analyzed by flow cytometry (10,000 events per sample) and data were expressed as mean fluorescence intensity (MFI).

Flow cytometry was employed to examine the immunophenotype of lymphocytes found in peripheral blood. The process involved obtaining a whole blood sample, which was then treated with a set of monoclonal human antibodies comprising anti-CD4 BV421, anti-CD3 PerCp, anti-CD8 BV605, anti-CD19 FITC, anti-CD45 Alexa Fluor 700, anti-CD56 BV650, anti-CD16 BV650, anti-TLR-2 APC, anti-TLR-4 PE, anti-TLR-7 PE, anti-TLR-8 APC, anti-TLR-3 PE, and anti-TLR-9 APC (Biolegend, San Diego, CA, United States). Following the antibody staining, a lysing buffer (BD, Franklin Lakes, NJ, United States) was applied to eliminate red blood cells, and the resulting cells were thoroughly washed and evaluated using a CytoFLEX LX instrument (Beckman Coulter, Indianapolis, IN, United States). Subsequently, data analysis was carried out utilizing the Kaluza Analysis program (sample analysis in Figure 1—CLL and Figure 2—CVID) The CytoFLEX LX flow cytometer was internally quality controlled using CytoFLEX Ready to Use Daily QC Fluorosphere reagents (Beckman Coulter, Indianapolis, IN, United States).