Quantifying Monocyte TLR2/4 Expression

Cells were fixed by diluting cells in a staining buffer (600 μL) and Inside Fix reagent (750 μL) from the Inside Stain Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were then centrifuged and washed in staining buffer (300 μL), and kept overnight at 4 °C. After another centrifugation, Inside Perm reagent (50 µL) from the Inside Stain Kit was added to pellets. Next, cells were incubated with conjugated antibody CD282 (TLR2)-APC and CD284-PE (3 µg per 10⁶ cells) (Miltenyi Biotec). Samples were finally washed and fixed in order to perform analysis using flow cytometer CytoFLEX S (Beckman Coulter Life Sciences, Indianapolis, IN, USA). CytExpert software (Beckman Coulter Life Sciences, Indianapolis, IN, USA) was used to process data on the basis of the monocyte population gated by FSC/SSC parameters [35 (link)].