Facscalibur

Single cell suspensions were stained immediately for activation markers or stimulated for 5 hours with 100 ng/ml PMA (Alexis Biochemical) and 1 µg/ml ionomycin (Calbiochem) in brefeldin A (BD Biosciences) for intracellular cytokine staining. Cells were treated with anti-FcγR (BioXCell) before staining with combinations of the following antibodies: anti-β1 Pacific Blue, anti-β7 FITC, anti-TCRvα2 allophycocyanin, anti-CD90.1 peridinin chlorophyll protein, anti-CD45.2 phycoerythrin (PE), anti-CD90.2 FITC, anti-IFN-γ PE, anti-TNF-α PE-cy7, anti-CD25 PE, anti-CD44 PE or Pacific Blue, anti-CD62L FITC (Biolegend), anti-CD3ε allophycocyanin, anti-α4 PE (BD Biosciences), anti-CD4 Qdot605 and a LIVE/DEAD dead cell stain kit (Invitrogen). The efficacy of all antibodies used in this study was confirmed extensively in vitro and compared to isotype control antibodies. For cytokine staining, cells were permeabilized using a Cytofix/Cytoperm Plus Kit following manufacturer’s instructions (BD Biosciences). Cell number was determined with AccuCheck Counting Beads (Invitrogen). Flow cytometry data were collected on a modified FACSCalibur (Cytek Development) or an LSRII (BD Biosciences) and analyzed using FlowJo (Tree Star).

Human 293F suspension cell cultures, maintained in FreeStyle 293 Expression Medium, were transiently transfected with the modified pVRC8400 expression vectors into which full-length HAs were cloned (Schmidt et al., 2013 (link)) using polyethylenimine (PEI). Transfection complexes were prepared in Opti-MEM and added to cells. At 14–18 hours post transfection cells were harvested for staining. Cells were pelleted at 300 ×g and washed with phosphate buffered saline (PBS) pH 7.4 supplemented with 1% BSA (FACS buffer). 50,000 cells were then incubated for one hour at room temperature with primary antibody at a concentration of 0.5 μg/ml in FACS buffer. Cells were washed three times in FACS buffer and incubated with 0.5 manufacturer’s specified reactions of a mouse anti-human IgG antibody conjugated with BB515 (clone G18–145, BD Horizon™ Cat#564581 ) Cells were washed thre times and then fixed in 2% paraformaldehyde (PFA). Fluorescence was measured using a FACSCalibur instrument (Cytek Development, Freemont, CA) and analyzed using FlowJo software (FlowJo Inc, Ashland, OR).

Flow cytometry was conducted on a Cytek modified FACSCalibur™ using the 488 blue, 637 red, and 407 violet lasers to excite active caspase-3 (PE), emission collected with a 580/20 filter, γH2AX (AlexaFluor 647) with a 661/16 filter, and DAPI with a 450/50 filter, respectively. 10,000 cells were collected per sample at low pressure. Single stain controls were used for compensation and gating was determined through unstained and fluorescence minus one controls (FMOs) (22 ), which are experimental cells stained with all the fluorophores minus the one fluorophore for which positive and negative cutoffs are determined. Gating and analysis were performed using the FCS Express 6 (DeNovo Software).