Alexa fluor 647 goat anti human igg h l cross adsorbed secondary antibody

HEK293T cells expressing HSV-1 gD as a surface antigen and non-transfected HEK293T cells (control) were washed twice with PBS. Cells (200,000/well) were added to a 96 well V bottom plate (USA Scientific). gD-specific antibodies were diluted to 20 μg/mL and serially diluted four-fold in PBS + 1% BSA before being added to the cells. After a 1-hour incubation on ice, the cells were washed twice with PBS + 1% BSA and stained with 10 μg/mL Alexa Fluor^™ 647 Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody (ThermoFisher Scientific) diluted in PBS with 1% BSA. After a 30 min incubation in the dark, cells were washed twice with PBS + 1% BSA and were resuspended in 100 μL of PBS prior to fixation with 4% paraformaldehyde. The antibody binding was measured by checking signal intensity of Alexa Fluor 647 using a MACSQuant Analyzer (Miltenyi) (Figure S2B). The experiment had two biological replicates. The data was analysed using FlowJo version 10.8.2.

HEK293T cells expressing HSV-1 gD as a surface antigen and non-transfected HEK293T cells (control) were washed twice with PBS. Cells (200,000/well) were added to a 96 well V bottom plate (USA Scientific). gD-specific antibodies were diluted to 20 μg/mL and serially diluted 4-fold in PBS +1% BSA before being added to the cells. After a 1-h incubation on ice, the cells were washed twice with PBS +1% BSA and stained with 10 μg/mL Alexa Fluor 647 Goat anti-Human IgG (H + L) Cross-Adsorbed Secondary Antibody (ThermoFisher Scientific) diluted in PBS with 1% BSA. After a 30 min incubation in the dark, cells were washed twice with PBS +1% BSA and were resuspended in 100 μL of PBS prior to fixation with 4% paraformaldehyde. The antibody binding was measured by checking signal intensity of Alexa Fluor 647 using a MACSQuant Analyzer (Miltenyi) (Figure S2B). The experiment had two biological replicates. The data was analyzed using FlowJo version 10.8.2.