The CellSimple™ Cell Growth Signaling Multiplex Bead Assay Kit is a bead-based 4-plex antibody assay founded upon the sandwich immunoassay principle. The assay kit allows for the simultaneous detection of four signaling molecules that are involved in the regulation of cell growth and survival. Each target-specific capture antibody has been covalently linked to a specific sized magnetic bead. Each kit contains enough reagents to run 50 samples allowing the user to generate 200 data points. The sample is incubated with the multiplex bead cocktail followed by a biotinylated detection antibody cocktail. Streptavidin linked Phycoerythrin is then used as a reporter to visualize the bound detection antibody. Analysis is then performed using the CellSimple™ Cell Analyzer, with the Multiplex Bead Assay Application.
CellSimple™ Cell Growth Signaling Multiplex Bead Assay Kit detects the target proteins Phospho-PRAS40 (Thr246), Phospho-S6 Ribosomal Protein (Ser235/236), Phospho-Akt (Ser473), and Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204). No significant cross-reactivity has been observed between targets. This kit is optimized for cell lysates diluted to a total protein concentration between 0.1 and 0.5 mg/mL (see kit protocol).
The CellSimple™ Cell Analyzer is a benchtop instrument that utilizes a disposable thin-film cassette and a combination of a 488 nm laser, two photomultiplier tubes (525/45 nm and 561 nm LP filters), Coulter Principle-based cell measurements, and on-board software to provide easy-to-run applications and data analysis. Data acquisition occurs within approximately 10 seconds per test. The instrument relies on disposable cassettes for sample handling, which alleviates the need for flow cell cleaning and fluidics maintenance and the instrument is small enough to be portable between the lab bench and the hood. Applications include quantitative assessments of cell viability, apoptosis, other labeled antibody markers and single and multiplexed bead-based assays for protein and cellular analysis.
The fundamental cellular behaviors of growth, proliferation and survival are controlled by a number of signal transduction pathways, in particular MAPK/Erk, PI3 Kinase, and mTOR. These pathways can be activated in response to a wide range of extracellular signaling cues such as growth factors, cytokines, hormones and neurotransmitters. MAPK/Erk is activated by dual phosphorylation at Thr202 and Tyr204 by both MEK1 and MEK2 (1,2). Akt is a protein kinase generally activated downstream of the PI3 kinase signaling pathway by phosphorylation at Ser473 and Thr308, by TORC2 complex and PDK1, respectively. These phosphorylation events are reliable predictors of Akt activation (3). Phosphorylation of PRAS40 at Thr246 by Akt relieves PRAS40 inhibition of TORC1 (3). mTOR is an important signaling hub that is a major component of two macromolecular complexes, TORC1 and TORC2. mTOR integrates growth factor signaling and nutrient availability, thus playing an important role in cell growth and homeostasis (4). mTORC1 phosphorylates p70 S6 Kinase at Thr389, leading to kinase activation and cell cycle progression. The S6 ribosomal protein is found downstream of p70 S6 kinase, and phosphorylation at Ser235/236 reflects mTOR pathway activation and predicts cell cycle progression (5).
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