Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays.
J Vis Exp
; (156)2020 02 21.
Article
em En
| MEDLINE
| ID: mdl-32150162
ABSTRACT
Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample's impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Bioensaio
/
Receptores Histamínicos
/
Transdução de Sinais
/
Histamina
/
Impedância Elétrica
/
Glioma
Limite:
Humans
Idioma:
En
Revista:
J Vis Exp
Ano de publicação:
2020
Tipo de documento:
Article