Cd19 pacific blue

Cell lysates (100 000 cell equivalents per well) were probed with antibodies to GAPDH (R&D Systems), β-actin (Sigma-Aldrich), and ACE2 (Novus Biologicals). The appropriate secondary horseradish peroxidase antibodies were used for imaging with an iBright 1500 system (Invitrogen), and ImageJ (Version 1.49 23) software was used for quantification. Cell surface expression was measured by means of flow cytometry using ACE2–phycoerythrin (PE) (Novus Biologicals), as recommended by the manufacturer. Primary cell populations were incubated with CD19-PacificBlue (BioLegend), CD3–fluorescein isothiocyanate (FITC), CD4-FITC, CD8-FITC, CD14– allophycocyanin (APC), and CD56-AlexaFluor700 (all BD Biosciences). Primary cells were treated with human immunoglobulin G (10 µg/1 million cells) to block Fc receptors on B cells. Data were acquired on an LSR II flow cytometer using a single-stained AbC bead kit (ThermoFisher) for compensation [17 (link)]. Viable cells were gated based on forward and side scatter, doublets were excluded, and “fluorescence minus 1” controls were assessed. PE quantitation beads (BD Quantibrite) were analyzed, according to the manufacturer’s instructions, to quantify ACE2 molecules per cell. At least 10 000 total events were collected in each experiment and the FlowJo program (Tree Star) was used for data analysis.