Cellomics HCS Reagent Kit for detection of phosphorylated AKT (Protein Kinase B).

control	treat cells	detect phospho–AKT	analyze	conclude

Description

The Cellomics Phospho-AKT Activation Kit measures phosphorylation of the 1, 2 and 3 isoforms of AKT, a key kinase involved in regulation of cell proliferation. The kit contains a polyclonal rabbit phospho-AKT antibody, a goat anti-rabbit secondary antibody conjugated to DyLight 649 Fluorophore, Whole Cell Stain Green, and the various other reagents and buffers required for immunofluorescent detection of phospho-AKT for high-content screening (HCS) assays.

AKT is activated through the PI3 kinase pathway by growth factors, cytokines, mitogens, and hormones. Once the signal has been transduced through the membrane by receptor tyrosine kinases, PI3 kinase phosphorylates AKT at serine473 and threonine308. Once phosphorylated, activated AKT phosphorylates key proteins involved in metabolism, protein synthesis, apoptosis, transcription factor regulation, and the cell cycle, including MDM2, FOXO, BAD, GSK-3b, and mTOR. Alterations in AKT signaling lead to uncontrolled cell proliferation, and genetic mutations in the PI3 kinase signaling pathway are prominent in colon, breast, and prostate cancers.

AKT activation was measured using IGF-1 treated 3T3 L1 pre-adipocytes and the assay was optimized with the Cellomics ArrayScan HCS Reader and Morphology Explorer BioApplication Software Module. In this application, many cell features, including texture measurements and intensity are available for quantitation. After stimulation with IGF-1, AKT is phosphorylated, resulting in activation and movement of AKT to the membrane periphery as well as other subcellular locations in the cytoplasm (Figure 1). Therefore, AKT activation may be quantitated using total cellular fluorescent intensity or changes in intracellular texture (including intracellular spots). Cells labeled by this kit may also be imaged using fluorescence or confocal microscopy.

	Nuclei (Hoechst dye)	Whole Cell Stain Green	Phospho-AKT (red)
Non-treated
Treated with IGF-1
Figure 1. AKT activation in 3T3 L1 pre-adipocytes treated with IGF-1. Cells were treated for 10 minutes with IGF-1 in media containing no glucose or serum after 24 hour growth factor starvation to measure AKT activation. Cells were fixed and stained with Hoechst 33342 dye for nuclei (blue), Whole Cell Stain Green, and phospho-AKT primary antibody plus DyLight 649 Goat Anti-Rabbit Secondary Antibody (red). Top panels are images from non-treated cells, and bottom panels are treated cells. Columns are nuclei (blue), whole cell (green) and phospho-AKT (red) fluorescent channels, respectively; hyperlinked images are color overlays of phospho-AKT plus nuclei or whole cell channels. Stimulation results in the activation of AKT and increased cytoplasmic staining.

Activation of AKT was measured in response to IGF-1 and PDGF treatment (Figure 2). An intracellular AKT intensity texture measurement (entropy intensity change) was used for quantitation. Left Panel: triplicate dose response curves of 3T3 L1 cells treated for 10 minutes with IGF-1. Right Panel: dose response curve of NIH 3T3 fibroblasts stimulated for 15 minutes with PDGF. The intracellular AKT intensity (cell average intensity) was used for quantitation.

Figure 2. Dose response curves of phospho-AKT in 3T3 L1 cells treated with IGF-1 (left) and NIH 3T3 fibroblasts treated with PDGF (right). Each data point represents eight wells. Curves in left panel represent separate data from three replicate plates. Single curve in right panel represents data from a single plate. Wells were scored using the output parameters of entropy intensity change in the left panel, and the cell average intensity in right panel. EC50 = 0.03+/-0.01 µg/ml IGF-1 and 40 ng/ml PDGF

Related Poster Presentation

	Kay Opperman, Krishna Vattem, and Richik N. Ghosh (2008) High-Content Analysis of Multiple Key Proteins in the AKT Signaling Pathway.
	Kay Opperman (2007). A new way to study the PI3-kinase signaling pathway using the AKT isoforms 1, 2 and 3. Pierce Previews 11(3):16-18.

Kit Contents

Ordering Information

Screening size kits and components (bulk and custom) available with special pricing. Please call.

To order Cellomics HCS Reagent Kits, please call 800-874-3723 in the U.S.
or contact your local distributor of Thermo Scientific Products.

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