Quantifying Surface Receptor Expression in Transfected Cells

Transiently transfected HEK293T cells were seeded in a poly-L-lysine coated 96-wells plate for 48 h at 37 °C and 5% CO₂. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% NP-40 as described above. In the case of determination of surface receptor expression levels, cells were only fixed with 4% paraformaldehyde. Receptor expression was detected using a Rat anti-HA antibody (1:1000 in 1% (v/v) FBS/PBS, Clone 3F10, Roche) and HRP-conjugated Goat-anti-Rat antibody (1:1000 in 1% (v/v) FBS/PBS, Pierce). Detection of intrabody-FLAG expression was done using a Mouse-anti-FLAG antibody (1:1000 in 1% (v/v) FBS/PBS, Clone M2, Sigma-Aldrich) and HRP-conjugated Goat-anti-Mouse antibody (1:2000 in 1% (v/v) FBS/PBS, Pierce). In the case of U251-iUS28 cells, receptor expression was detected using an Rabbit-anti-US28 antibody (1:1000 in 1% (v/v) FBS/PBS, Covance^{24 (link)}) and HRP-conjugated Goat-anti-Rabbit antibody (1:1000 in 1% (v/v) FBS/PBS, Bio-Rad). Incubation with antibodies was done for 1 h at RT. Wells were washed three times with 1× PBS in between all incubation steps. Antibody binding was detected using 1-Step ultra TMB-ELISA substrate (Thermo Fisher Scientific) and the reaction was stopped with 1 M H₂SO₄. Optical density was measured at 450 nm with a PowerWave plate reader (BioTek). Data were analyzed using GraphPad Prism version 8.0.