We have expressed various human and rat Transporters in mammalian cell lines. GVK BIO has extensive experience in screening compounds against Transporters. Some of the Transporters validated by us are:
- Norepinephrine Transporter-NAT (Human)
- Dopamine Transporter-DAT (Human) -3
- Serotonin Transporter-5HT (Human)
- Sodium-dependent Glucose Co-transporter (Human)
- Peptide Transporter (Human)
- Sodium/Phosphate Transporter (Human)
To screen compounds against transporters, we perform the following assays:
- Radioligand uptake assay (See more)
The uptake of specific substrates by the transporters over-expressed in mammalian cells is determined. Cells are incubated with radiolabelled substrates at specific temperature. Excess substrate is removed by washing and the cells are lysed. The radioactivity of the released substrate is measured. We have validated uptake assay for NaPi, SGLT transporters.
- Membrane potential assay (See more)
Proton movement due to uptake of substrates by transporters causes intracellular acidification and membrane depolarization. This change in membrane potential is measured by fluorescent dye using FLIPR. We have validated Membrane potential assay for PEPT transporters.
- Calcein AM uptake assay (See more)
At GVK BIO calcein AM assay is employed specifically for efflux transporters. Calcein AM assay is a fluorescent based assay, where intracellular esterases cleaves calcein AM to give fluorescent calcein, which is measured.
Cells are incubated with calcein AM dye and with or without inhibitors. Accumulation of free fluorescent calcein is measured using Envision. We have validated calcein AM uptake assay for various efflux transporters viz., ABC transporters.