Cd15 pb

Absolute counts of peripheral blood leukocytes were determined for all study participants using BD Trucount tubes, six-color TBNK Reagent and, in addition, CD123 BUV395 (BD Biosciences), CD15 PB, CD193 BV605, and HLA-DR BV785 (Biolegend), and CD14 PE-Cy5 (eBioscience) according to the manufacturer’s instructions. Samples were fixed in 1× BD fluorescence-activated cell sorter (FACS) lysing solution (BD Biosciences) for 2 h and acquired on a BD FACSymphony A5 instrument, equipped with ultraviolet (355nm), violet (405 nm), blue (488 nm), yellow/green (561 nm), and red (637 nm) lasers. For absolute cell count calculations, the number of events for populations of interest was divided by the number of bead events and multiplied by the BD Trucount bead count.

For the flow cytometry, the cells were washed with PBA (PBS supplemented with 0.5% bovine serum albumin (BSA), 1% FBS, and 0.1% sodium azide), and incubated with conjugated antibodies against surface markers (CD3-CF (Immunostep, Salamanca, Spain); CD56-PC5 (Phycoerythrincyanin 5), CD16-PE-Cy7 (phycoerythrin-Cy7), CD14-APC (Allophycocyanin), CD15-PB (Pacific Blue), and CXCR3-APC (BioLegend, San Diego, California, USA) at 4 °C for 30 minutes in the dark. For intracellular staining, after surface labelling, the cells were fixed with 1% p-formaldehyde for 10 min at room temperature (RT), permeabilized with 0.2% saponin for 10 min at RT, and stained with anti-CXCL10-PE (BD, San Jose, California, USA) or mouse IgG2a-PE (Beckman Coulter, Brea, California, USA) for 30 min. The cells were washed in PBA and analyzed using a Gallios Flow Cytometer (Beckman Coulter, Brea, California, USA). The analysis of the experiments was performed using either Kaluza (Beckman Coulter, Brea, California, USA) or FlowJo software (Ashland, Oregon, USA, https://www.flowjo.com).