Use of primary cells is preferred in drug research as they lack immortalizing mutations and are not monoclonal. Therefore, primary cells predict drug responses more reliably. Upon activation of the drug relevant G-protein coupled receptors, second messengers become active such as calcium ions or cAMP. Two assays that report on these second messengers and are amenable to primary cells are presented here.
The cytosomal release of Ca2+ was measured in 96 well plates with the CloneticsTM calcium sensor that was transiently transfected into human umbilical vein endothelial cells (HUVECs). The cells were stimulated by injection of histamine. The GloSensor cAMP sensor was transiently transfected into human mesenchymal stem cells to display cAMP generation upon varying doses of Formeterol, PGE1 or Forskolin. The cAMP assay was performed in 96- and 384-well plates.
The luminescence plate readers by BMG LABTECH reliably detected the fast changes in Ca2+ and determined the EC50 values of cAMP-stimulating agents in primary cells.