Muse® Assays for Cell Signaling Analysis

Current methods for monitoring cell signaling, such as Western blotting, ELISA, bead-based assays and flow cytometry, all have advantages and limitations. Population analyses (such as Westerns, ELISAs or bead-based assays) are quantitative, but do not allow discrimination at the single cell level, masking true response. Traditional flow cytometry can provide highly quantitative data on single cells, but expertise, costly instrumentation or access to a core facility may stand in the way of routine use in signaling research. The Muse® Cell Analyzer provides the highly quantitative, reproducible, single cell-level results of flow cytometry in a compact, benchtop platform requiring little sample preparation and minimal expertise.

Activation Dual Detection Kits

These simple, yet precise, assays for study cell signaling pathways each include a pair of carefully optimized antibodies that bind to the same protein: one to detect total protein expression and another to detect the phos-phorylated form of the same target. By using two-parameter analysis, the Muse® instrument delivers target-specific detection of phosphorylation while eliminat-ing false positives and enhancing the signal-to- noise ratio. These kits also contain all the necessary fixation, permea-bilization, and assay buffers to provide complete solutions for signaling analysis.

Data generated include:

  • Percentage of inactivated cells
  • Percentage of activated cells (via phosphorylation)
  • Percentage of non-expressing cells

Choose from multiple Muse® kits to assess the activation of key cell signaling pathways:

  • Muse® H2A.X Activation Dual Detection Kit (Cat. No. MCH200101)
  • Muse® EGFR-RTK Activation Dual Detection Kit (Cat. No. MCH200102)
  • Muse® PI3K Activation Dual Detection Kit (Cat. No. MCH200103)
  • Muse® MAPK Activation Dual Detection Kit (Cat. No. MCH200104)
  • Muse® Bcl-2 Activation Dual Detection Kit (Cat. No. MCH200105)
  • Muse® PI3K/MAPK Activation Dual Detection Kit (Cat. No, MCH200108)
  • Muse® Multi Color DNA Damage Kit (H2A.X/ATM)(Cat No. MCH200110)

We are continually releasing new Muse® assay modules and kits. Please visit www.emdmilllipore.com/muse for the most up-to- date listing of Muse® Assays. New assay software modules can be downloaded free of charge from the website.

Featured Kits

Muse® H2A.X Activation Dual Detection Assay (Cat. No. MCH200101)

This assay allows the researcher to monitor and accurately measure phospho-specific Histone H2A.X activation in a population of cells. The histone H2A.X resides downstream of the DNA damage kinase signaling cascade. As the level of DNA damage increases, the level of phospho Histone H2A.X (also known as γH2A.X) increases, accumulating at the sites of DNA damage. This accumulation of phospho Histone H2A.X is often used to indicate the level of DNA damage in the cell.

The Muse™ H2A.X Activation Dual Detection Kit includes two directly conjugated antibodies, a phospho-specific anti-phospho- Histone H2A.X (Ser139)-Alexa Fluor®555 and an anti-Histone H2A.X-PECy5 conjugated antibody to measure total levels of Histone H2A.X. This two-color kit is designed to detect the extent of Histone H2A.X pathway activation by measuring H2A.X phosphorylation relative to the total H2A.X expression in any given cell population.

Data output for the Muse® H2A.X Activation Dual Detection Kit. HeLa cells were exposed to 10 µM Etoposide for 24 hours to induce DNA damage, stained with both anti-phospho- Histone H2A.X (Ser139) and anti-Histone H2A.X antibodies in multiplex and analyzed using the Muse® Cell Analyzer. Summary data and statistics (not shown) show the relative percentages for each population as it is calculated within the total cell population. Dot plot and bar graph data are shown here. Cells that express H2A.X correspond to the data in the top two quadrants of the dot plot (inactivated and activated, representing about 99.6% of the total cell population). But of this cell population, 90.8% is activated upon treatment, indicating that DNA damage is present. By presentation of both data sets, the total: phospho ratio can be determined.

Muse® PI3K Activation Dual Detection Kit (Cat. No. MCH200103)

Akt/PKB is a Ser/Thr kinase and a major known effector of the PI3 Kinase pathway. It is involved in multiple signaling pathways regulating metabolism, apoptosis, cell cycle control, angiogenesis, differentiation, cell growth, proliferation and more. The Muse® PI3K Activation Dual Detection Kit includes two directly conjugated antibodies, a phospho-specific anti-phospho- Akt (Ser473), Alexa Fluor®555 and an anti-Akt, PECy5 conjugated antibody to measure total levels of Akt. Use this two-color kit to measure the extent of Akt phosphorylation relative to the total Akt expression at a single-cell level.

Data output for Muse® PI3K Kinase Activation Dual Detection Assay. In Jurkat cells, Akt is constitutively activated. Jurkat cells were exposed to 1 µM wortmannin for 60 minutes at 37°C to inhibit the Akt signaling cascade response, fixed, per-meabilized, and then stained with both anti- phospho- Akt (Ser473) and anti-Akt/PKB antibodies in multiplex. Samples were analyzed using the Muse® Cell Analyzer. The statistics captured in this assay show the relative percentages for each sub-population as it is calculated within the total cell population. Cells which express Akt correspond to the top two quadrants of the dot plot (81.2% of the total cell population). But of this cell population, 69.4% is deactivated upon treatment, at-tenuating the constitutive activation of the Akt signaling pathway. By presentation of both data sets, the total: phospho ratio can be determined.

Muse® Multi-Color DNA Damage Kit (Cat. No. MCH200107)

This kit provides a quick and easy way to detect the activation of ATM and H2A.X by flow analysis. The kit includes two directly conjugated antibodies, a phospho-specific ATM (Ser1981)-PE and a phospho-specific Histone H2A.X-PECy5 conjugated antibody to measure the extent of DNA damage. Both antibodies are carefully titrated and optimized together to ensure maximal performance when run in multiplex, alleviating the need for any additional optimization. Two-color analysis of ATM and Histone H2A.X activation in multiplex provides more reliable DNA damage detection than measuring either protein on its own. This kit also contains optimized fixation, permeabilization, and assay buffers to providea complete solution for DNA damage signaling analysis.

Data generated include:

  • Percentage of negative cells (no DNA damage)
  • Percentage of ATM-activated cells
  • Percentage of H2A.X-activated cells
  • Percentage of DNA double-strand breaks (dual activation of both ATM and H2A.X)

Data output for Muse® Multi-Color DNA Damage Kit. HeLa cells were exposed to 10 µM Etoposide for 24 hours to induce DNA damage, and then stained with both anti-phospho- Histone H2A.X (Ser139) and anti-phospho- ATM (Ser1981) antibodies in multiplex. Samples were acquired using the Muse® Cell Analyzer. The statistics captured in this assay show the relative percentages for each population as it is calculated within the total cell population (top). Cells which express ATM, H2A.X, or both can be seen by the data on upper left, lower right, and upper right quadrants of the dot plot, respectively (bottom). In this cell population, 64.5% shows co-activation of ATM and H2A.X upon treatment, indicating DNA damage and double-strand breaks are present.

     
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