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Kinasera-TRF live cell assay
ARC-Lum probes can be used for monitoring the activity of protein kinases in live cells. The assay can be performed in the 96-well microplate format with 40-100 microL detection volumes. Attached cells are treated with ARC-Lum probe for 60 minutes to enable the uptake of the probe. The cells are washed to remove the excess of the probe and nutrient mixture. The change in the luminescence signal upon stimulation or inhibition of the cells can be monitored in real time using conventional plate reader.
Monitoring the activity of PKA in live cells. CHO cells over-expressing PKA (A) and in HEK293 cells with endogenous expression of PKA (B) were grown to 80% confluence on black 96-well cell culture plates, incubated with ARC-Lum probe for 60 minutes in serum free Ham's F-12 nutrient mixture, washed twice with HBSS. The luminescence intensity of intracellular ARC-Lum probe was monitored using PheraStar plate-reader (BMG) with HTRF module [TRF excitation filter (BMG 337), emission at 675(50) nm; 30 flashes, delay 50 ?s, integration time 150 ?s] in 100 ?L of HBSS. The increase in luminescence signal was obtained upon intracellular activation of PKA either via direct stimulation of cell-permeable adenylate cyclase activator forskolin (A: ?) or via activation of ?-adrenergic receptors by isoproterenol (B: ?, ?). Both stimulation pathways lead to the activation of PKA and the formation of the complex of ARC-Lum probe and PKAc. TRF signal in unstimulated cells (A: ?, B: ?) remains unchanged. TRF signal is significantly reduced upon the addition of H89, a cell permeable inhibitor of PKA (A: ?, B: ?, ?) as the ARC-Lum probe is displaced from its complex with PKAc.