Pe anti mouse cd47 antibody

Fifty μL of peripheral blood was collected in Eppendorf tubes containing 0.5M EDTA to prevent coagulation. Blood samples were incubated with Fc block for 10 m on ice (TruStain FcX™, Biolegend, Cat# 101320) followed by a cocktail of antibodies (1:100 dilution) for 30 min on ice; APC/Cyanine7 anti-mouse CD45 Antibody (Biolegend, 103116, clone 30-F11), Brilliant Violet^® 711 Anti-CD11b Rat Monoclonal Antibody (Biolegend, Cat#101242, clone: M1/70), Alexa Fluor^® 700Anti-Ly-6C Rat Monoclonal Antibody (Biolegend, Cat#128024, clone: HK1.4), PerCP/Cyanine5.5 anti-mouse CXCR2 Antibody (Biolegend, Cat#149307), PE anti-mouse CD47 Antibody (Biolegend, Cat#127507), Alexa Fluor^® 594 anti-mouse CEACAM1a Antibody (Biolegend, Cat#134522), Zombie NIR™ Fixable Viability Kit (Biolegend, Cat#423106), and APC anti-mouse CD54 Antibody (Biolegend, Cat#116119). After 30 min, all samples were incubated with BD Phosflow™ Lyse/Fix Buffer (BD Biosciences, 558049) following manufacturer’s protocol. Following lyse/Fix, cells were washed twice with 1× PBS containing 2% FBS, and resuspended in FACS buffer containing 2% FBS in PBS. Samples were run in Aurora Spectral Flow Cytometer (Cytek) and data analysis was done using Flowjo version 8.8.7 software.

In order to confirm the chimeric gC (cgC) expression from the recombinant virus and that the expressed cgC is anchored on cell surface, 293 cells were infected with 1 pfu/cell of either FusOn-CD47-Luc or FusOn-Luc. Cells were harvested 24 h later and stained either with 5% PE anti-mouse CD47 antibody (BioLegend, San Diego, CA, USA) or first with 1% mouse anti-HA tag and then with 2% goat anti-mouse -FITC antibody (Sigma, St. Louis, MO, USA) in 2% FBS-PBS at 4° C for 30 min. Cells stained with either PE Rat IgG or FITC goat anti-mouse IgG as the isotype control. The stained cells in 2% FBS-PBS were analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA).