Fcs express 7

Uninfected VERO E6 cells were subjected to the full inactivation protocol (S1). Cells were then stained with 10uM of calcein green (Invitrogen, C3100MP) and incubated for 20 minutes at room temperature. Flow cytometry data were then collected and analyzed using instrument BD Accuri C6 and FCSexpress 7 software (Version 7.12.0007, respectively).

Flow Cytometry was performed through the UCI Stem Cell Research Center Flow Cytometry Core using the BD Fortessa. 200,000 cells were processed for each of the samples, and data was analyzed using FCS Express 7. Plots were designed displaying 50,000 events each and reflect identical fluorescent compensation values.

The phenotype of the CD14+ cells was assessed at Day 0 right after isolation and on Day 7. To collect the adherent cells on Day 7, the cells were washed once with PBS and then covered with 1 mL 2.5 mM EDTA and refrigerated on ice for 1 hour. The cells were labeled with monoclonal antibodies (mAbs) from the Monoclonal Antibody Center at Washington State University (https://vmp.vetmed.wsu.edu/resources/monoclonal-antibody-center, Pullman, WA). The panel consisted of a negative control, and mAbs specific for CD14 (CAM36A), CD163 (LND68A), CD172a (DH59B), CD209 (209MD26A), and CD205 (ILA53A). All mAbs were developed in mice. Briefly, the cells were washed and then suspended in first wash buffer and divided into 15 mL conical tubes in 100 µL aliquots. The cells were incubated for 15 minutes with 50 µL each of the primary monoclonal antibodies as previously specified (Davis et al., 2007 (link)). The cells were washed and then incubated with 100 µL of the goat anti-mouse secondary antibody Alexa Fluor® 488 (Thermo Fisher Scientific, MA) for 15 minutes. The cells were then washed once with second wash buffer, resuspended in 2% PBS buffered formaldehyde, transferred to flow tubes, and stored at 4°C until flow cytometry was performed. Samples were run on a BD FACS Calibur™ and analyzed using FCS Express 7.