Cd66b percpcy5

Whole blood was lysed by diluting in lysis buffer (1x) (Biolegend) and washed in cold FACS buffer to retrieve a leukocyte pellet. Cell populations were distinguished by fluorescently labelling using a previously optimised polychromatic staining panel; LIVE/DEAD® Fixable Blue Dead Cell Stain Kit (Invitrogen), CD18 PECy5, CD66b PERCPCy5.5, CD14 BV421, HLA DR BV510, CD15 605, CD20 AF700, CD19 APC Cy7, HLA DM PE, CD3 PECF594, and HLA ABC (BD Biosciences). Relevant fluorescence minus one (FMO) controls and negative controls were used to identify positively stained populations. Lymphocytes, monocytes and granulocytes were identified by size forward scattered light (FSC)/ side scattered light (SC]) and then further phenotyped into live CD3− CD14+ monocytes, CD3+ lymphocytes, CD3− CD19/CD20 expressing B cells and CD66b/CD15 positive neutrophils. Flow cytometry was performed on the Fortessa instrument (BD Biosciences) and data analysed using ‘Flow Jo’ (Tree Star) software.

After incubation at 37°C, 5% CO₂, cells were incubated at 4°C for 15 minutes, stained with fluorochrome conjugated monoclonal antibodies, and suspended in saline. Antibodies were: CD16 PE, CD62L-PECy7 and CD66b-PerCPCy5.5 (BD Pharmingen, San Diego, CA, USA). Flow cytometry data (at least 50,000 events per sample) were acquired using either a FACSVerse Flowcytometer (BD Bioscience) or a FACS CantoII (BD Bioscience). Data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).
Neutrophils were identified by forward- and side-scatter characteristics and, in some cases, CD16+ expression. Infected or bystander population of neutrophils were identified as CFSE+ or CFSE- cells, respectively (S1 Fig).
As the experiments with healthy controls subjects were performed at the laboratory at Complexo Hospitalar Universitário Professor Edgard Santos in Salvador city, we used a different flow cytometry than we have at the laboratory in the endemic area.

Antibodies from BD Biosciences; CD15-BV650 (#563142, clone W6D3), CD45-PE-CF594 (#562279 clone HI30), CD16-V450 (#560474 clone 3G8), CD62L-BV510 (#563203 clone DREG-56), CD47-PE (#556046 clone B6H12), CD36-APC (#550956 clone CB38), CD11b-APC-Cy7 (#557754 clone ICRF44), CD66b-PerCP-Cy5.5 (#562254 clone G10F5), CD49d-BV711(#563177 clone 9F10), CD43-BB515 (#564542 clone 1G10) and CD24-PE-Cy7 (#561646 clone ML5).