Pe conjugated mouse anti human igg fc antibody

Flow cytometry-based Cell-ELISA (CELISA) binding of mAbs with HCoV spikes was performed as described previously.⁵² (link)^,104 (link) A total of 4x10⁶ HEK293T cells were seeded into 10cm round cell culture dishes and incubated at 37°C. After 24h, HEK293T cells were transfected with plasmids encoding full-length HCoV spikes and were incubated for 36-48h at 37°C. The cells were harvested and distributed into 96-well round-bottom tissue culture plates for individual staining reactions. For each staining reaction, cells were washed three times with 200μl FACS buffer (1xPBS, 2%FBS, 1mM EDTA). The cells were stained for 1h on ice in 50μl staining buffer with 10μg/ml of primary antibody. After washing three times with 200μl FACS buffer, the cells were stained with 50μl/well of 1:200 diluted R-phycoerythrin (PE)-conjugated mouse anti-human IgG Fc antibody (SouthernBiotech Cat# 9040-09) and 1:1000 dilution of Zombie-NIR viability dye (BioLegend Cat# 423105) on ice in dark for 45min. Following three washes with FACS buffer, the cells were resuspended and analyzed by flow cytometry (BD Lyrics cytometer), and the binding data were generated by calculating the Mean Fluorescence Intensity using FlowJo 10 software. Mock-transfected 293T cells were used as a negative control.

Binding of monoclonal antibody to various human coronavirus (HCoV) spike proteins expressed on the surface of HEK293T cells was determined by flow cytometry, as described previously (85 (link)). Briefly, HEK293T cells were transfected with different plasmids encoding full-length HCoV spike proteins and were incubated for 36 to 48 hours at 37°C. Post incubation cells were trypsinized to prepare a single cell suspension and were distributed into 96-well plates. Monoclonal antibodies were prepared as 5-fold serial titrations in FACS buffer (1x phosphate-buffered saline (PBS), 2% fetal bovine serum (FBS), 1 mM EDTA), starting at 10 μg/ml, 6 dilutions. 50 μl/well of the diluted samples were added into the cells and incubated on ice for 1 hour. The plates were washed twice in FACS buffer and stained with 50 μl/well of 1:200 dilution of R-phycoerythrin (PE)-conjugated mouse anti-human IgG Fc antibody (SouthernBiotech cat.# 9040–09) and 1:1000 dilution of Zombie-NIR viability dye (BioLegend cat.# 423105) on ice in dark for 45 minutes. After another two washes, stained cells were analyzed using flow cytometry (BD Lyrics cytometers), and the binding data were generated by calculating the percent (%) PE-positive cells for antigen binding using FlowJo 10 software.