Cd11b fluorescein isothiocyanate fitc

Fresh, anticoagulated (K₂‐EDTA), whole blood (100 μL) was mixed with fluorescent‐conjugated monoclonal antibodies specific to CD11b‐fluorescein isothiocyanate (FITC; Biolegend, San Diego, CA) and CD66b‐phycoerythrin (PE; BD Biosciences, San Jose, CA). Samples were mixed and incubated for 15 min in the dark, after which the samples were lysed with 2 mL of 1× FACS lysing solution (BD Biosciences), mixed and incubated in the dark for an additional 8 min. Following incubation, samples were centrifuged at 300g for 8 min and washed with 2 mL of 1× wash buffer containing 1% fetal bovine serum (FBS) in a 1× phosphate‐buffered saline (PBS) solution. Samples were centrifuged again at 300g for 8 min, and the supernatant was removed. Samples were then fixed in 300 μL of 2% paraformaldehyde in PBS.

Three micrograms per milliliter RVFV Gn protein (custom; GenScript)-coated MaxiSorp plates (Thermo Fisher) were blocked with 5% BSA in PBST (0.01%) for 1 h at 37°C. MAbs were added to wells at 5 μg/ml and incubated for 2 h at 37°C. NK cells were isolated by negative selection from C57BL/6 mouse spleens using EasySep mouse NK cell isolation kit (StemCell Technologies). Purified NK cells were added at 2 × 10⁵ cells/well in the presence of brefeldin A (Sigma-Aldrich), GolgiStop (BD), and anti-CD107a conjugated to phycoerythrin (PE) (BioLegend clone 1D4B) to wells already containing Gn/MAb. NK cells were incubated for 5 h at 37°C. Cells were then washed and stained with near-infrared (IR) fluorescent reactive dye (Thermo Fisher). Cells were stained for cell surface markers CD3 allophycocyanin (APC)-Cy7 (BioLegend clone 17A2), CD11b fluorescein isothiocyanate (FITC) (BioLegend clone M1/70), and NK1.1 APC (BioLegend clone PK136). The purity of NK cells was confirmed by CD3 APC (BioLegend clone 17A2), CD19 BV421 (BioLegend clone 6D5), NKp46 PE-Cy7 (Biolegend clone 29A1.4), and CD14 APC-Cy7 (BioLegend clone Sa14-2) staining. All cells were fixed in BD Cytofix/Cytoperm and then analyzed by flow cytometry on a BD LSRFortessa flow cytometer. All flow cytometric data were analyzed using FlowJo 10.7.1.