Cd16 bv786

Human neutrophils were infected with Y. pestis KIM6+ or S. aureus as described above in wells of 96 well plates. Additionally, neutrophils were left uninfected/untreated or stimulated with Phorbol 12-myristate 13-acetate (PMA, 1 μM, Sigma, St. Louis, MO), E. coli lipopolysaccharide (LPS, 1 μg/ml, Sigma), or N-formyl-Met-Leu-Phe (fMLP, 10 μM, Sigma) where indicated as positive controls for neutrophil activation. Cells were harvested from wells at 15 min and 4 h post-infection and stained for flow cytometry with a cocktail of fluorescently labeled antibodies against the following markers: CD88-PerCP/Cy5.5 (BioLegend, San Diego, CA), CD66b-Alexa647, CD62L-PETR, CD16-BV786, CD63-PECy7, CD64-Alexa700, CD11b-Alexa488, and CD54-BV421 (all from BD Immunocytometry, San Jose, CA). The fixable viability dye efluor780 (ThermoFisher) was also included and analysis was limited to live, efluor780^low/negative cells. After staining, cells were washed with 2% FBS in PBS and resuspended in 2% paraformaldehyde in PBS prior to analysis on a LSR-II flow cytometer (BD Immunocytometry). Data were analyzed using FlowJo 10.2 software.

Nonexpanded donor-matched cells before and after use of the STEM device (n = 6) were stained using antibodies for MSCs and macrophage markers, including CD90-Alexa700 (AbD Serotec), CD45-V450, CD14-FITIC, CD16-BV786, HLA-DR-PE, M2 marker CD206-APC, and M1 marker CD86-PECy7 (all from BD Biosciences).^{14 (link)}
Isotype controls were used for each antibody with nonspecific antibody binding blockade using phosphate-buffered saline containing 10% mouse serum and 1% human IgG. For all flow cytometry analyses, gating was first established (at 1% positivity) on the isotype controls and then applied to the corresponding markers. Data were acquired and analyzed using Cytoflex S (Beckham Coulter).