Zombie violet viability stain

Viral protein expression in B cells was analyzed by flow cytometry using a BD LSRFortessa flow cytometer (BD Biosciences). Vaccine was detected through GFP expression and wild-type cells were detected using MeV hemagglutinin (H) protein by antibody staining using mouse anti-H (MilliporeSigma, MAB8905, clone CV1/CV4) and anti-mouse IgG fluorescein isothiocyanate (FITC) conjugated secondary antibody (eBioscience, 11-4010-82). Immunophenotyping of B cells was performed using phycoerythrin conjugated anti-CD27 (BD Pharmigen, 555441), allophycocyanin conjugated anti-CD19 (BD Pharmigen, 555415), BD Horizon Brilliant Ultraviolet (BUV) 395 conjugated anti-CD20 (BD Horizon, 563782), Alexa 700 conjugated anti-IgD (BD Pharmigen, 561302), BD Brilliant Violet (BV) 605 conjugated anti-CD24 (BD Horizon, 562788), BV786 conjugated anti-CD38 (BD Horizon, 563964), and Zombie Violet viability stain (Biolegend, 423113). Positive fluorescent values were determined as signal above the isotype control for each fluorophore. Positive values for viral fluorescence were determined as the signal above cells stained for viral protein expression at 0 h.

4T1 and BEND3 cells were separately cultured in RPMI 1640 with 10% FBS (Gibco 11875093) at 37 °C at 5% CO₂. Cells were dissociated with StemPro Accutase (Gibco A1110501). Cells were stained with ZombieViolet viability stain (BioLegend 423113) in HEPES buffer (20 mM HEPES, 150 mM NaCl, pH 7.4). Fluorescent nanoparticles were incubated with cells in HEPES buffer with 1% w/v BSA for 30 min on ice. Cells were washed three times with HEPES-1% BSA prior to examination on an Attune NxT Flow Cytometer (Invitrogen). Data were analyzed using FlowJo.