Anti tlr4 apc clone hta125

Freshly isolated neutrophils (1 × 10⁵) were stained with 2 μg mL⁻¹ anti-CXCR1-FITC (R&D Systems; clone 42075), 3 μg mL⁻¹ anti-CXCR2-FITC (R&D Systems; clone 48311) or 5 μg mL⁻¹ anti-TLR4-APC (clone HTA125; eBioscience, Hatfield, UK) antibodies or their relevant concentration-matched isotype controls for 20 min on ice. Postincubation, cells were washed once in PBS/1% BSA, resuspended in 100 μL PBS and transferred to polypropylene FACS tubes. Flow cytometry was conducted using a CyAn_ADP™ bench-top cytometer (Dako, Cambridgeshire, UK) with 5000–10 000 neutrophils acquired for analysis, which was performed using Summit v4.3 software (Dako Colorado Incorporation, Fort Collins, CO, USA). The percentage of antigen-positive neutrophils was recorded along with the corresponding median fluorescence intensity (MFI) values.

The highly purified basophils of all donors were resuspended in stimulation buffer (IMDM, Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (all from Gibco^®, Life Technologies, Carlsbad, CA, USA), and 2.5 ng/ml IL-3 (Sigma-Aldrich). 40.000 cells in 100 μl were used per reaction. Basophils were incubated for 30 minutes at 4°C with anti-TLR1-PE (clone GD2.F4), anti-TLR2-eFluor450 (clone T2.5), anti-TLR4-APC (clone HTA125), and anti-TLR6-Biotin (clone h-Per6) (20 minutes followed by 10 minutes Streptavidin-APC-eFluor780 treatment) or the respective isotype controls (eBioscience, San Diego, CA, USA and ImmunoTools, Friesoythe, Germany). Specificity of the anti-TLR antibodies has previously been demonstrated[11 (link)–14 (link)]. The cells were measured on a FACS Canto II (BD, Franklin Lakes, NJ, USA).