Anti cd14 antibody

Neutrophils were isolated from fresh venous blood collected in lithium heparin vacutainers (BD, Scoresby, Australia) using the EasySep Direct Human Neutrophil Isolation Kit (STEMCELL Technologies, Tullamarine, Australia) as per manufacturer’s instructions. Neutrophil purity was assessed by cell morphology using light microscopy of Giemsa stained smears of the isolated cells, and viability was assessed using trypan blue exclusion. Monocytes were isolated from both fresh venous blood, collected in lithium heparin vacutainers (BD), as well as from buffy coats supplied from the Australian Red Cross Blood Service. Monocytes were isolated by negative selection using the RosetteSep Human Monocyte Enrichment Cocktail (STEMCELL Technologies) as per manufacturer’s instructions. Monocyte purity was assessed by staining (anti-CD14 antibody, BioLegend, San Diego, CA) and measuring CD14⁺ cells by flow cytometry. Monocytes were either frozen in fetal bovine serum (FBS) in 20% dimethyl sulfoxide (DMSO) in liquid nitrogen for later use or used immediately after isolation. Natural killer (NK) cells were isolated from fresh venous blood collected in sodium heparin vacutainers (BD). NK cells were isolated by negative selection using the RosetteSep Human NK Enrichment Cocktail (STEMCELL Technologies) as per manufacturer’s instructions.

In order to access cell surface expression of TLR4, Wildtype, Tlr4-deficient or Tlr5-deficient BMDM were harvested, washed with PBS and were exposed to ultrapure LPS for 0,15,30, 60 or 90 min. Cells were washed with cold PBS and gently lifted from the culture dishes using a cell lifter. Cells numbers were estimated and cells were aliquoted in 1 × 10⁶ cells per tube in the FACS buffer (0.5% BSA, 0.1% NaN3, and 2 mM EDTA in PBS). Cells were blocked for 20 min on ice in a blocking solution (FACS buffer, 10% species specific serum, and 1% FCR block). Cells were stained using APC anti-mouse CD284 (TLR4) Antibody (clone SA15-21), anti-CD14 Antibody (Biolegend) or isotype controls for 30 min on ice. Cells were washed two times with 1 mL FACS buffer after staining, suspended in 500 uL FACS buffer containing 1 mg/mL propidium iodide (to identify dead cells) and analyzed on a BD FACSAria II equipment.