lumGFP plasmid DNA has been described previously (Blum et al., 2000 (link)). Cells were nucleofected with 20mg of plasmid per reaction (around 1x106 cells per reaction). For lumGFP experiments, cells were analysed 24h after nucleofection.
lumGFP secretion collection was performed by washing and incubating confluent cells in SF for 60 minutes. Cells were then lysed to determine total lumGFP levels. Relative amounts of GFP were determined by sandwich ELISA using MaxiSorp plates (Thermo Fisher Scientific) coated with sheep anti-GFP (BioRad), followed by blocking then incubation with samples. Plates were washed and incubated with rabbit anti-GFP (Invitogen) followed by washing and a final incubation with goat anti-Rabbit conjugated with horseradish peroxidase (HRP) (Jackson Laboratories). Plates were developed with o-phenylenediamine dihydrochloride and hydrogen peroxide in a citrate phosphate buffer. Absorbance was analysed at 450 nm in a Thermomax microplate reader (Molecular Devices) using a kinetic protocol with a reading every 30 s for 30 min. A standard curve was made using a lumGFP nucleofected lysates serially diluted. Results are shown as % of secreted GFP from the total GFP measured in each sample (secreted plus lysate).
lumGFP secretion collection was performed by washing and incubating confluent cells in SF for 60 minutes. Cells were then lysed to determine total lumGFP levels. Relative amounts of GFP were determined by sandwich ELISA using MaxiSorp plates (Thermo Fisher Scientific) coated with sheep anti-GFP (BioRad), followed by blocking then incubation with samples. Plates were washed and incubated with rabbit anti-GFP (Invitogen) followed by washing and a final incubation with goat anti-Rabbit conjugated with horseradish peroxidase (HRP) (Jackson Laboratories). Plates were developed with o-phenylenediamine dihydrochloride and hydrogen peroxide in a citrate phosphate buffer. Absorbance was analysed at 450 nm in a Thermomax microplate reader (Molecular Devices) using a kinetic protocol with a reading every 30 s for 30 min. A standard curve was made using a lumGFP nucleofected lysates serially diluted. Results are shown as % of secreted GFP from the total GFP measured in each sample (secreted plus lysate).