Alexa fluor 488 conjugated goat anti mouse antibody

GST-tagged recombinant N-terminal domains of human CEACAM1 were purified and utilized as previously described (47 (link)). Gc was grown overnight with various IPTG concentrations, and 1 × 10⁸ bacteria were incubated with GST-tagged N-CEACAM1 for 30 minutes at 37°C with end-over-end rotation. Gc was washed and stained to detect the presence of CEACAM with a mouse anti-GST antibody p1A12 (BioLegend 640802) followed by an Alexa Fluor 488 conjugated goat anti-mouse antibody (Jackson ImmunoResearch 115-545-164). Gc was then resuspended in 2% paraformaldehyde containing DAPI DNA stain. To process and analyze N-CEACAM1 binding, ImageStream X Mk II imaging flow cytometer with INSPIRE and IDEAS v. 6.2 software was used. In each replicate, single-color Gc was used to create the respective data set’s compensation matrix. Bacteria were gated by DAPI positivity, singlet size, focused images, and Alexa Fluor 488 positivity.

Chromosome spreading was performed as described previously [87 (link)]. The slides carrying the spreads were treated with appropriate primary and secondary antibody combinations prior to fluorescence microscopy. The primary antibodies, mouse anti-Myc (1:300), rat anti-HA (3F10; 1:300), and mouse anti-GFP (1:300), were purchased from Roche (Germany). AlexaFluor488-conjugated goat anti-mouse antibody (1:200) and TRITC-conjugated goat anti-rat antibody (1:200) were obtained from Jackson ImmunoResearch Laboratories.

Neutralizing antibody levels were measured by the Broad Institute using a live-virus SARS-CoV-2 PRNT as previously described (34 (link)). Neutralizing antibody serum samples were tested at a 1:40 dilution and then serially diluted 4-fold up to 4 times before being mixed with live SARS-CoV-2 (D614G) for 1 hour. The mixture was added to Vero E6-TMPRSS2 cells for 48 hours, and then, infected cells were detected with anti–SARS-CoV-2 nucleoprotein mouse primary antibody (Sino Biological) and a secondary Alexa Fluor 488–conjugated goat anti-mouse antibody (Jackson ImmunoResearch).