FlowCellect™ PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection kit

FlowCellect™ PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection kit

FlowCellect

25 Tests / Kit

Recent evidence suggests that cross-talk between the PI3K and MAPK signaling pathways exist. We use pAkt and pERK antibodies to examine the PI3K/MAPK interactions. Additionally, to further interrogate the interplay between these two pathways, cell proliferative marker Ki-67 is used to validate the final biological effect.

Millipore’s FlowCellect™ PI3K/MAPK Dual Activation and Cancer Detection Kit is designed to examine this cross-talk in a multi-parametric fashion by providing three fully validated and optimized antibody biomarkers to measure specific cell signaling events in flow applications. The three antibodies provided in the kit are Anti-phospho-Akt Alexa Fluor 488 conjugate, Anti-phospho-ERK R-Phycoerythrin conjugate, and Anti-Ki-67 PerCP conjugate. By utilizing all three antibody biomarkers simultaneously in flow applications, we now have the ability to thoroughly evaluate the “cross-talk” between PI3K and MAPK pathways and to further determine the consequence of their interplay in cell proliferation and differentiation by measuring their effect on Ki-67 expression.

Phosphorylated Akt and phosphorylated ERK are included in the kit to provide the end user with the means to cross-examine both the PI3K and MAPK signaling pathways simultaneously. It has been suggested that the phosphorylation of Akt can result in the inhibition, or dephosphorylation, of phospho-Raf on Ser 259 [Jun, T. et al. (1999) ]. By inactivating Raf, this will essentially block the MAPK signaling pathway resulting in an inactivated phospho-ERK. In some situations, a surface receptor (such as IGF-1) will activate both the PI3K and MAPK pathways leading to the phosphorylation of both Akt and ERK. However, since this interaction or “cross-talk” exist between the two pathways, it is critical to investigate their interactions in both a spatial and temporal manner.

In a recent study, we have examined the effects of IGF-1 activation by the addition of Insulin on both the PI3K and MAPK signaling pathways on HEK293 cells. We have performed this experiment implementing critical time points for signaling evaluation: activation at 3 minute and at 5 minute time intervals. The resulting responses indicate that cross-talk is observed, noted by a sharp decrease in ERK expression. This transient response is attributed to the phosphorylation of Akt, which in turn will shut off phosphorylated ERK as noted above.

In order to validate the biological effect of phospho-protein activation by a given stimulus, cell cycle marker Ki-67 is used since it is a true measurement of the “proliferative fraction”. Ki-67 is present in all phases of the cell cycle except for G0. However, Ki-67 can only be detected in flow cytometry when cells are going through M phase as Ki-67 expression patterns are punctate in all other phases producing weaker signals. But by using a cell cycle arrest reagent, Cell Cycle Stop™, cell proliferation measured by Ki-67 expression can be accurately determined as cells are arrested at M phase and are clearly visible in flow cytometry analysis. In order to accurately measure the cell proliferative activity by Ki-67 expression, however, cells must be treated for at least 12 hours with a combination of Cell Cycle Stop™ and a given cell stimulus to fully achieve enough circulating cells to be captured in M-phase. Additionally, prior to cell treatments all cultures must be serum starved for 24 hours to essentially reset the cell cycle and bring most circulating cells back to G0 [Littleton, RJ. et al. (1991) ].

Using multi-parametric flow analysis, we are able to cross-examine these signaling events and their biological consequence simultaneously, providing a biological correlation between pathway activation and cancer proliferation.

Examination of cell signaling pathways and monitoring their activation status have been extremely important for researchers to understand the detailed mechanisms of cellular functions and the cause of various diseases. Many signal transduction pathways have been implicated to lead to multiple outcomes such as apoptosis, cell differentiation, cell growth and cell proliferation, all of which have been extensively studied for the treatment of various cancers and autoimmune diseases.

The study of cell signaling pathways are now made easier with the use of activation status-specific and phospho-specific antibodies. Measurement of protein phosphorylation with phospho-specific antibodies has given insight into kinase signaling cascades [Krutzik, P.O. et al. (2003) ]. Multi-parameter phospho flow cytometry is a powerful tool for studying multiple pathways in a mixed cell population at the same time.

Much excitement in the field of signal transduction has centered on the discovery of increasing cross-talk among signaling pathways [Jun, T. et al. (1999) ]. Recent evidence has suggested that communication between the PI3K and MAPK pathways exist downstream from the cell surface [Jun, T. et al. (1999), Moelling, K. et al. (2002), Zimmermann, S. et al. (1999)]. The ability for signaling pathways to cross-talk adds an extra dimension and complexity when evaluating pathways of interest. Since signal transduction pathways are an elaborate highway of events, the ability to monitor these key intracellular “checkpoints” simultaneously provides researchers a very powerful tool for analyzing complicated cell events such as cancer cell proliferation by measuring the activity of multiple cell signaling pathways.

Millipore’s FlowCellect™ PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection kit is designed to allow the researcher to cross examine both the PI3K and MAPK signaling pathways, as well as cell proliferation simultaneously. This kit provides three directly conjugated antibodies which are optimized for multi-color flow cytometry applications for the detection of Akt phosphorylation, ERK1/2 phosphorylation and Ki-67 cancer marker expression. Ki-67 has been indicated to be a reliable tumor proliferative marker in cancer cells [Ishikuro, A. et al. (1997)]. The fraction of Ki-67 positive cells, often defined as the “proliferative fraction”, has prognostic value in many tumors [Darzynkiewicz, Z. et al. (2001)].

Although the use of phospho-specific antibody staining as a biomarker may give some measure of target activation, it may not necessarily correlate with the desired biological effect (e.g. growth inhibition or apoptosis). Researchers would likely benefit from the inclusion of both phospho-specific signal transduction markers (such as pERK and pAkt) and a proliferation marker (such as Ki-67) to allow a true measure of treatment evaluation at the level of the tumor [Smalley, KSM et al. (2007)].

All FlowCellect™ kits are optimized on the bench-top Guava® flow cytometry systems, which saves valuable time and sample volume. All kits contain optimized fixation, permeabilization, wash and flow buffers to provide researchers with a complete solution for simultaneous detection of multiple pathway activations. With the Guava platform and FlowCellect™ kits, one can finally have an easy, reliable and fully validated solution to study the complex cell signaling pathways right in the comfort of your own lab.

Flow Cytometry

Human

• Alexa Fluor® is a registered trademark of Molecular Probes, Inc.
• Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Lot to lot in-process testing on all kit components

• 1. 20X Anti-phospho-Erk1/2 (Thr202/Tyr204, Thr185/Tyr187) R-Phycoerythrin conjugate Monoclonal Antibody: (Part No. CS203329) One 150 µL vial
• 2. 20X Anti-phospho-Akt1/PKBα (Ser473) Alexa Fluor 488 conjugate Monoclonal Antibody: (Part No. CS203326) One 150 µL vial
• 3. 20X Anti-KI-67 PerCP conjugate Monoclonal Antibody: (Part No. CS203324) One 150 µL vial
• 4. Fixation Buffer: (Part No. CS202122) One 13 mL bottle
• 5. 10X Wash Buffer: (Part No. CS202123) One 13 mL bottle
• 6. 5X Assay Buffer: (Part No. CS202124) One 55 mL bottle
• 7. 1X Permeabilization Buffer: (Part No. CS202125) One 13 mL bottle
• 8. Cell Cycle Stop™: (Part No. CS203335) One vial supplied with 1 mg

Mouse

4-8°C for antibodies and buffers

• ¹²⁵I
• Fluorescent

FlowCellect™ PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection kit

Flow Cellect

ExpressPro

Monoclonal Antibody

Research