Acuri c6 plus flow cytometer

Cellular uptake by BMDMs in the presence or absence of immunized mice sera or naive sera was compared using CPMV-Cy5, eCPMV-Cy5, and PEG-eCPMV-Cy5 particles. Prior to use, BMDMs were removed from M-CSF and resuspended in serum-free DMEM media. CPMV-Cy5, eCPMV-Cy5, or PEG-eCPMV-Cy5 was preincubated with immunized sera or naive sera (1:200 dilutions) or media only (1 μg virus/50 μL serum-free DMEM/well) in a 96-well v-bottom plate at room temperature for 20 min. BMDMs were then added to the wells (200 000 cells/50 μL serum-free DMEM/well), and the plate was incubated at 37 °C and 5% CO₂ for 30 min. Postincubation, cells were washed twice with cold PBS containing 1 mM EDTA and resuspended in staining buffer (PBS with 2% (v/v) FBS, 1 mM EDTA, 0.1% (w/v) sodium azide). Next, Fc receptors were blocked using anti-mouse CD16/CD32 antibody (BioLegend) for 15 min and then stained with fluorescently labeled antibodies FITC-CD11b (clone M1/70) (BioLegend) and PE-F4/80 (clone BM8) (BioLegend) for 30 min on ice. Poststaining cells were washed twice and immediately analyzed using a BD Acuri C6 Plus flow cytometer. Data were analyzed using FlowJo v8.6.3 software.