Pe mouse anti human cd16

PE-Cy7 Mouse anti-Human CD14 (BD), PE Mouse Anti-Human CD16 (BD), APC Rat anti-human TLR-9 (BD), FITC Mouse Anti-Human CD64 (BD), APC Cy7 anti-Human CD206 (Biolegend, San Diego, CA, USA), and PerCP-Cy5.5 anti-Human CD192 (CCR2) (Biolegend).

For phenotyping, the peripheral blood mononuclear cells (PBMCs) (1 × 10⁶) were labeled with antibodies and fluorochromes specific for CD14 (APC Mouse Anti-Human CD14; BD Biosciences) and CD16 (PE Mouse Anti-Human CD16; BD Biosciences). Samples were run on a FACScalibur flow cytometer (BD Biosciences, Mississauga, ON, Canada), using Cell Quest software, and analyzed using Infinicyt software, version 1.7 (Cytognos, Salamanca, Spain). Briefly, peripheral blood cells (PBCs) were labeled and incubated for 30 min at room temperature in the dark. After this, 500 ul of PBS was added and centrifuged (5 min at 1500 rpm) for washing, and the supernatant discarded. The cells were resuspended in 500 ul saline solution, and immediately acquired in a flow cytometer.

CSC populations (CD24⁻/CD44⁺ and ALDH⁺) were measured by flow-cytometry of single-cell suspensions obtained from tumor biopsies (Additional file 1: Supplemental Materials) by using anti-human CD44-APC (clone G44-26) and CD24-FITC (clone ML5, RUO) antibodies (BD Biosciences, Franklin Lakes, NJ), and ALDEFLUOR assay (StemCell Technologies, Inc., Vancouver, BC, Canada), respectively, as previously published [13 (link)].
Additional analyses were directed to determine CXCR1 (PerCP/Cy5.5 mouse anti-human CD181 (CXCR1; BioLegend, San Diego, CA) and cell lineages using the following antibodies: APC mouse anti-human CD44 (BD Bioscience, San Jose, CA; catalog #559942), PE-Cy7 mouse anti-human CD24 (#561646), PE mouse anti-human CD2 (#555327), PE mouse anti-human CD31 (#555446), PE mouse anti-human CD3 (#555333), PE mouse anti-human CD18 (#555924), PE mouse anti-human CD16 (#555407), PE mouse anti-human CD19 (#555413), and PE mouse anti-human CD45 (#555483).