Cxcr1 pe

Panel 1 included antibodies allowing the gating of PMN-MDSCs in the PBMCs and the analysis of expression of surface markers. It comprised of CD3-BV510, CD56-BV510, CD19-BV510, CD11b-APCH7, CD15-BV711, CD14-BV650, HLA-DR-BV605, CD10-PE-CF594 (Becton Dickinson), CD16-AF700 (BioLegend, Ozyme, Saint-Cyr-l’Ecole, France), CD33-PC7 (eBiosciences, Thermo Fisher), LIVE/DEAD fixable aqua (Life Technologies, Villebon-sur-Yvette, France). LOX-1-PE, CD11b-activated-PE (clone CBRM1/5) (Bio-Legend, Ozyme), CD62L-PE, CD66b-PE, PDL-1-BV421, CXCR1-PE, CD172ab-AF647 (BD Biosciences) were alternately included in Panel 1.
Panel 2 was used for functional analysis. It contained CD15-FITC (eBiosciences, Thermo Fisher), CD3-PC5 (BD Biosciences) and LIVE/DEAD near-IR (Live Technologies,). CD66b-PE, CD10-PE-CF594 and CD16-AF700 were occasionally added. PBMCs were stained 20 min at room temperature with fluorescent reagents pre-mixed in PBS, washed, then fixed with 4% paraformaldehyde and analyzed by FACS within 4 days on LSRII-SORP cytometer (BD Biosciences) equipped with four lasers (405 nm/100 mW, 488 nm/100 mW, 560 nm/50 mW and 630 nm/40 mW). PMT were set using unstained and fully stained samples. Compensations were performed with beads stained with corresponding reagents. Data were exported and analyzed with FlowJo (version 9-2, MacOS X).

One hundred thousand purified neutrophils were stained with the following antibodies: CD15 VioBlue (dilution: 1:100, clone: VIMC6, Cat. No.: 130–113-488, Miltenyi Biotec), CD16 FITC (dilution: 1:100, clone: REA423, Cat. No.: 130–113-392, Miltenyi Biotec), fMLP receptor Alexa Fluor 647 (final dilution: 1:100, clone: 5F1, Cat. No.: 565623, BD Biosciences, San Jose, CA), CXCR1 PE (dilution: 1:100, clone: 8F1, Cat. No.: 130–105-352, Miltenyi Biotec), and CXCR2 PE-Vio770 (dilution: 1:20, clone: REA208, Cat. No.: 130–100-930, Miltenyi Biotec). After an incubation step of 15 min in the dark at 4 °C, the suspensions were diluted 1:1 with PBS and analyzed on a MACSQuant VYB (Miltenyi Biotec).