The CellSimple™ Apoptosis Multiplex Bead Assay Kit is a four-plex bead-based 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 stress and apoptosis. 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 done using the CellSimple™ Cell Analyzer with the Multiplex Bead Assay Application.
All components in this kit are stable for at least 6 months when stored at the recommended temperature. Kit should be stored at 4°C with the exception of Lysis Buffer, which is stored at –20°C (packaged separately).
CellSimple™ Apoptosis Multiplex Bead Assay Kit detects the target proteins Cleaved PARP (Asp214), Cleaved Caspase-3 (Asp175), Cleaved Caspase-7 (Asp198) and Phospho-Bad (Ser112). 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.
Cell death can occur due to a variety of circumstances including nutrient deprivation, inability to generate or store the energy required for metabolic functions, or deleterious environment that causes irreparable damage. Cells integrate multiple signals from a variety of sources before following either pro- or anti-apoptotic pathways (1). These signals can often carry conflicting information. Assessing the net effect of these processes in cell populations can be achieved by monitoring changes in a number of key signaling components. The caspase-3 and caspase-7 proteases exert a pro-apoptotic function through cleavage of multiple cellular targets. Caspase-3 and caspase-7 are activated by cleavage at Asp175 and Asp198, respectively (2). PARP is a DNA repair and apoptosis enzyme that is inactivated by cleavage at Asp214 by caspase-3 or caspase-7 (3). Akt is activated by stimulation of growth-factor receptors and primarily promotes anabolic growth and survival signals via targeting its broad array of substrates. Akt phosphorylates Bad at Ser112 and inhibits its ability to induce apoptosis (4).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. CellSimple is a trademark of Cell Signaling Technology, Inc.