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.
Muse® signaling assays were developed to give researchers a rapid, simple method for detecting the activation of key signaling pathways in cell populations. Many of these assays rely on antibodies that detect both total and activated targets, enabling the automated calculation of the ratio of activated to total target.
Cell Signalling Assays
Muse® H2A.X Activation Dual Detection Assay Kit
(Cat. No.
MCH200101)
Muse® Activation Dual Detection kits include a pair of antibodies that bind to the same protein; one to detect total protein expression and another to detect the phosphorylated form of the same target. Using two parameter analysis, we can achieve target specific detection of phosphorylation and eliminate false positives while enhancing the signal to noise ratio. Data generated using the Muse® Cell Analyzer with the Muse® software provides:
- Percentage of inactivated cells
- Percentage of activated cells (via phosphorylation)
- Percentage of non-expressing cells
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, resulting in a normalized and accurate measurement of H2A.X activation after stimulation. Simultaneous measurement of both total and phospho-Histone H2A.X confirms target specificity of the phosphorylation event. Together, a total and phospho antibody duo performed in multiplex provides an enhanced and more reliable detection of the phospho: total ratio within a mixed cell population. Using this antibody pair provides a sensitive and valuable tool to study the factors that induce DNA damage and/or affect DNA repair, and allow one to explore the linkage between DNA damage, cell cycle checkpoints, and initiation of apoptosis.
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Kit Components
- 20X Anti-phospho-Histone H2A.X (Ser139), Alexa Fluor 555: (Part No. CS208203). One vial containing 250 µL
- 20X Anti-H2A.X, PECy5: (Part No. CS208202). One vial containing 300 µL
- 5X Assay Buffer: (Part No. CS202124). One bottle containing 55 mL
- Fixation Buffer: (Part No. CS202122). One bottle containing 13 mL
- 1X Permeabilization Buffer: (Part No. CS203284). One bottle containing 14 mL
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Materials Recommended
- Test tubes for sample preparation and storage
- Tissue culture reagents, i.e. HBSS, PBS w/o Ca2+ or Mg2+, cell dislodging buffers, etc.
- Pipettes with corresponding tips capable of accurately measuring 10 – 1000 µL
- Tabletop centrifuge capable of achieving 300 x g
- Mechanical vortex
- Deionized Water (for buffer dilution)
- Cells of interest in suspension (e.g. Jurkat)
- Microcentrifuge tubes with screw caps, 1.5 mL (VWR, Cat. No. 16466-030, or equivalent)
- Muse® Cell Analyzer
- Muse® System Check Kit (Cat. No. MCH100101)
- Guava ICF Instrument Cleaning Fluid (Cat. No. 4200-0140), optional
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Expected Results
Figures A and B. 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-Histone H2A.X antibodies in multiplex. Samples were acquired using the Muse® Cell Analyzer and statistical results are shown above. Figure A shows the results summary, while Figure B shows results displayed by both dot plot and bar graph data.
The statistics captured in this assay show the relative percentages for each population as it is calculated within the total cell population. Cells which express H2A.X can be seen by the data on 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 DNA damage is present. By presentation of both datasets, one can now determine the total: phospho ratio within their testing samples.
Muse® EGFR-RTK Activation Dual Detection Assay Kit
(Cat. No.
MCH200102)
Muse® Activation Dual Detection kits include a pair of antibodies that bind to the same protein; one to detect total protein expression and another to detect the phosphorylated form of the same target. Using two parameter analysis, we can achieve target specific detection of phosphorylation and eliminate false positives while enhancing the signal to noise ratio. Data generated using the Muse® Cell Analyzer with the Muse® software provides:
- Percentage of inactivated cells
- Percentage of activated cells (via phosphorylation)
- Percentage of non-expressing cells
The Muse® EGFR-RTK Activation Dual Detection Kit includes two directly conjugated antibodies, a phospho-specific anti-phospho-EGFR (Tyr1173)-Alexa Fluor 555 and an anti-EGFR-PECy5 conjugated antibody to measure total levels of EGFR expression. This two color kit is designed to detect the extent of EGFR pathway activation by measuring EGFR phosphorylation relative to the total EGFR expression in any given cell population, resulting in a normalized and accurate measurement of EGFR activation after stimulation. Simultaneous measurement of both total and phospho-EGFR confirms target specificity of the phosphorylation event, and provides an enhanced and more reliable detection of the phospho: total ratio within a mixed cell population. Sufficient reagents are provided to perform 50 tests.
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Kit Components
- 20X Anti-phospho-EGFR (Tyr1173), Alexa Fluor 555: (Part No. CS208204). One vial containing 250 µL
- 20X Anti-EGFR, PECy5: (Part No. CS208205). One vial containing 300 µL
- 5X Assay Buffer: (Part No. CS202124). One bottle containing 55 mL
- Fixation Buffer: (Part No. CS202122). One bottle containing 13 mL
- Permeabilization Buffer: (Part No. CS202125). One bottle containing 13 mL
-
-
Materials Recommended
- Test tubes for sample preparation and storage
- Tissue culture reagents, i.e. HBSS, PBS w/o Ca2+ or Mg2+, cell dislodging buffers, etc.
- Pipettes with corresponding tips capable of accurately measuring 10 – 1000 µL
- Tabletop centrifuge capable of achieving 300 x g
- Mechanical vortex
- Deionized Water (for buffer dilution)
- Cells of interest in suspension (e.g. Jurkat)
- Microcentrifuge tubes with screw caps, 1.5 mL (VWR, Catalog No. 16466-030, or equivalent)
- Muse® Cell Analyzer
- Muse® System Check Kit (Catalog No. MCH100101)
- Guava ICF Instrument Cleaning Fluid (Catalog No. 4200-0140), optional
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Expected Results
Figures A and B. A431 cells were exposed to 100 ng/mL EGF for 5 minutes to induce an EGFR signaling cascade response, fixed, permeabilized, and then stained with both anti-phospho-EGFR (Tyr1173) and anti-EGFR antibodies in multiplex. Samples were acquired using the Muse® Cell Analyzer and statistical results are shown above. Figure A shows the results summary, while Figure B shows results displayed by both dot plot and bar graph data.
The statistics captured in this assay show the relative percentages for each population as it is calculated within the total cell population. Cells which express EGFR can be seen by the data on the top two quadrants of the dot plot (inactivated and activated, representing about 93% of the total cell population. But of this cell population, 91% is activated upon treatment, indicating activation of the EGFR signaling pathway is present. By presentation of both datasets, one can now determine the total: phospho ratio within their testing samples.
Muse® PI3K Activation Dual Detection Assay Kit
(Cat. No.
MCH200103)
Muse® Activation Dual Detection kits include a pair of antibodies that bind to the same protein; one to detect total protein expression and another to detect the phosphorylated form of the same target. Using two parameter analysis, we can achieve target specific detection of phosphorylation and eliminate false positives while enhancing the signal to noise ratio. Data generated using the Muse® Cell Analyzer with the Muse® software provides:
- Percentage of inactivated cells
- Percentage of activated cells (via phosphorylation)
- Percentage of non-expressing cells
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. This two color kit is designed to measure the extent of Akt phosphorylation relative to the total Akt expression in any given cell population. By doing such, the levels of both the total and phosphorylated protein can be measured simultaneously in the same cell, resulting in a normalized and accurate measurement of PI3K activation after stimulation. Moreover, simultaneous measurement of both total and phospho-Akt confirms target specificity of the phosphorylation event. Together, a total and phospho antibody duo performed in multiplex provides an enhanced and more reliable detection of the phospho: total ratio within a mixed population. Sufficient reagents are provided to perform 50 tests.
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Kit Components
- 20X Anti-phospho-Akt (Ser473), Alexa Fluor555: (Part No. CS208206). One vial containing 250 µL
- 20X Anti-Akt/PKB, PECy5: (Part No. CS208207). One vial containing 300 µL
- 5X Assay Buffer: (Part No. CS202124). One bottle containing 55 mL
- Fixation Buffer: (Part No. CS202122). One bottle containing 13 mL
- Permeabilization Buffer: (Part No. CS202125). One bottle containing 13 mL
-
-
Materials Recommended
- Test tubes for sample preparation and storage
- Tissue culture reagents, i.e. HBSS, PBS w/o Ca2+ or Mg2+, cell dislodging buffers, etc.
- Pipettes with corresponding tips capable of accurately measuring 10 – 1000 µL
- Tabletop centrifuge capable of achieving 300 x g
- Mechanical vortex
- Deionized Water (for buffer dilution)
- Cells of interest in suspension (e.g. Jurkat)
- Microcentrifuge tubes with screw caps, 1.5 mL (VWR, Catalog No. 16466-030, or equivalent)
- Muse® Cell Analyzer
- Muse® System Check Kit (Catalog No. MCH100101)
- Guava® ICF Instrument Cleaning Fluid (Catalog No. 4200-0140), optional
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Expected Results
Figures A and B. Jurkat cells were exposed to 1 µM wortmannin for 60 minutes at 37°C to inhibit the Akt signaling cascade response, fixed, permeabilized, and then stained with both anti-phospho- Akt (Ser473) and anti-Akt/PKB antibodies in multiplex. Samples were acquired using the Muse® Cell Analyzer and statistical results are shown above. Figure A shows the results summary, while Figure B shows results displayed by both dot plot and bar graph data.
The statistics captured in this assay show the relative percentages for each population as it is calculated within the total cell population. Cells which express Akt can be seen by the data on the top two quadrants of the dot plot (inactivated and activated, representing about 81.2% of the total cell population. But of this cell population, 69.4% is deactivated upon treatment, attenuating the constitutive activation of the Akt signaling pathway in jurkat cells. By presentation of both datasets, one can now determine the total: phospho ratio within their testing samples.
Muse® MAPK Activation Dual Detection Assay Kit
(Cat. No.
MCH200104)
Muse® Activation Dual Detection kits include a pair of antibodies that bind to the same protein; one to detect total protein expression and another to detect the phosphorylated form of the same target. Using two parameter analysis, we can achieve target specific detection of phosphorylation and eliminate false positives while enhancing the signal to noise ratio. Data generated using the Muse® Cell Analyzer with the Muse® software provides:
- Percentage of inactivated cells
- Percentage of activated cells (via phosphorylation)
- Percentage of non-expressing cells
The Muse® MAPK Activation Dual Detection Kit includes two directly conjugated antibodies, a phospho-specific anti-phospho-ERK1/2 (Thr202/Tyr204, Thr185/Tyr187)-Phycoerythrin and an anti-ERK1/2-PECy5 conjugated antibody to measure total levels of ERK. This two color kit is designed to measure the extent of MAPK phosphorylation relative to the total MAPK expression in any given cell population. By doing such, the levels of both the total and phosphorylated protein can be measured simultaneously in the same cell, resulting in a normalized and accurate measurement of MAPK activation after stimulation. Moreover, simultaneous measurement of both total and phospho-ERK1/2 confirms target specificity of the phosphorylation event. Together, a total and phospho antibody duo performed in multiplex provides an enhanced and more reliable detection of the phospho: total ratio within a mixed population. Sufficient reagents are provided to perform 50 tests.
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Kit Components
- 20X Anti-ERK1/2, PECy5: (Part No. CS208198). One vial containing 300 µL
- 5X Assay Buffer: (Part No. CS202124). One bottle containing 55 mL
- Fixation Buffer: (Part No. CS202122). One bottle containing 13 mL
- 1X Permeabilization Buffer: (Part No. CS203284). One bottle containing 14 mL
-
-
Materials Recommended
- Test tubes for sample preparation and storage
- Tissue culture reagents, i.e. HBSS, PBS w/o Ca2+ or Mg2+, cell dislodging buffers, etc.
- Pipettes with corresponding tips capable of accurately measuring 10 – 1000 µL
- Tabletop centrifuge capable of achieving 300 x g
- Mechanical vortex
- Deionized Water (for buffer dilution)
- Cells of interest in suspension (e.g. Jurkat)
- Microcentrifuge tubes with screw caps, 1.5 mL (VWR, Catalog No. 16466-030, or equivalent)
- Muse® Cell Analyzer
- Muse® System Check Kit (Catalog No. MCH100101)
- Guava ICF Instrument Cleaning Fluid (Catalog No. 4200-0140), optional
-
Expected Results
Figures A and B. Jurkat cells were exposed to 1 µM staurosporine for 5 hours to induce cell death and illicit a Bcl-2 signaling cascade response, the cells were then fixed, permeabilized, and stained with both anti-phospho-Bcl-2 (Ser70) and anti-Bcl-2 antibodies in multiplex. Samples were acquired using the Muse® Cell Analyzer and statistical results are shown above. Figure A shows the results summary, while Figure B shows results displayed by both dot plot and bar graph data. The statistics captured in this assay show the relative percentages for each population as it is calculated within the total cell population. Cells which express Bcl-2 can be seen by the data on the top two quadrants of the dot plot (inactivated and activated, representing about 95% of the total cell population). But of this cell population, 92% is dephosphorylated upon treatment as indicated by a left shift, confirming inactivation of the Bcl-2 signaling pathway upon treatment. By presentation of both datasets, one can now determine the total: phospho ratio within their testing samples.
Muse® Bcl-2 Activation Dual Detection Assay Kit
(Cat. No
MCH200105)Muse® Activation Dual Detection kits include a pair of antibodies that bind to the same protein; one to detect total protein expression and another to detect the phosphorylated form of the same target. Using two parameter analysis, we can achieve target specific detection of phosphorylation and eliminate false positives while enhancing the signal to noise ratio. Data generated using the Muse® Cell Analyzer with the Muse® software provides:
- Percentage of inactivated cells
- Percentage of activated cells (via phosphorylation)
- Percentage of non-expressing cells
The Muse® Bcl-2 Activation Dual Detection Kit includes two directly conjugated antibodies, a phospho-specific anti-phospho-Bcl-2 (Ser70)-Alexa Fluor 555 and an anti-Bcl-2-PECy5 conjugated antibody to measure total levels of Bcl-2 expression. This two color kit is designed to detect the extent of Bcl-2 pathway activation by measuring Bcl-2 phosphorylation relative to the total Bcl-2 expression in any given cell population. By doing such, the levels of both the total and phosphorylated protein can be measured simultaneously in the same cell, resulting in a normalized and accurate measurement of Bcl-2 activation after treatment. Moreover, simultaneous measurement of both total and phospho-Bcl-2 confirms target specificity of the phosphorylation event. Together, a total and phospho antibody duo performed in multiplex provides an enhanced and more reliable detection of the phospho: total ratio within a mixed cell population. Sufficient reagents are provided to perform 50 tests.
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Kit Components
- 20X Anti-phospho-Bcl-2 (Ser70), Alexa Fluor 555: (Part No. CS208208). One vial containing 250 uL
- 20X Anti-Bcl-2, PECy5: (Part No. CS208209). One vial containing 300 uL
- 5X Assay Buffer: (Part No. CS202124). One bottle containing 55 mL
- Fixation Buffer: (Part No. CS202122). One bottle containing 13 mL
- Permeabilization Buffer: (Part No. CS202125). One bottle containing 13 mL
-
-
Materials Recommended
- Test tubes for sample preparation and storage
- Tissue culture reagents, i.e. HBSS, PBS w/o Ca2+ or Mg2+, cell dislodging buffers, etc.
- Pipettes with corresponding tips capable of accurately measuring 10 – 1000 µL
- Tabletop centrifuge capable of achieving 300 x g
- Mechanical vortex
- Deionized Water (for buffer dilution)
- Cells of interest in suspension (e.g. Jurkat)
- Microcentrifuge tubes with screw caps, 1.5 mL (VWR, Catalog No. 16466-030, or equivalent)
- Muse® Cell Analyzer
- Muse® System Check Kit (Catalog No. MCH100101)
- Guava ICF Instrument Cleaning Fluid (Catalog No. 4200-0140), optional
-
Expected Results
Figures A and B. Jurkat cells were exposed to 1 µM staurosporine for 5 hours to induce cell death and illicit a Bcl-2 signaling cascade response, the cells were then fixed, permeabilized, and stained with both anti-phospho-Bcl-2 (Ser70) and anti-Bcl-2 antibodies in multiplex. Samples were acquired using the Muse® Cell Analyzer and statistical results are shown above. Figure A shows the results summary, while Figure B shows results displayed by both dot plot and bar graph data. The statistics captured in this assay show the relative percentages for each population as it is calculated within the total cell population. Cells which express Bcl-2 can be seen by the data on the top two quadrants of the dot plot (inactivated and activated, representing about 95% of the total cell population). But of this cell population, 92% is dephosphorylated upon treatment as indicated by a left shift, confirming inactivation of the Bcl-2 signaling pathway upon treatment. By presentation of both datasets, one can now determine the total: phospho ratio within their testing samples.
Muse® Multi-Color DNA Damage Kit
(Cat. No.
MCH200107)
This kit is designed to enable researchers a quick and easy way to detect the activation of ATM and H2A.X using the Muse® Cell Analyzer. The kit was designed and optimized using a DNA damaging reagent, Etoposide, on HeLa cells as a model system, but can also be used with other human cell lines to investigate both the physical and chemical factors which can induce the DNA damage response through the ATM dependent signaling pathway. Data generated using the Muse® Cell Analyzer along with the corresponding Muse® software module provides statistical values measuring:
- Percentage of negative cells (e.g. 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)
The Muse® Multi-Color DNA Damage 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 in testing samples. This two color kit is designed to detect the phosphorylation state of ATM and Histone H2A.X simultaneously by flow analysis. This kit contains optimized fixation, permeabilization, and assay buffers to provide researchers with a complete solution for DNA damage signaling analysis.
Muse® PI3K/MAPK Dual Pathway Activation Kit
(Cat. No.
MCH200108)
The Muse® PI3K/MAPK Dual Pathway Activation Kit is designed to enable researchers a quick and easy way to cross examine both the PI3K and MAPK signaling pathways simultaneously using the Muse® Cell Analyzer. Recent evidence suggests that there is cross-talk between the PI3K and the MAPK signaling pathways. Antibodies against phosphorylated Akt (pAkt) and phosphorylated ERK (pERK) can be used to examine PI3K/MAPK interactions. Data generated using the Muse® Cell Analyzer along with the corresponding Muse® software module provides statistical values measuring:
- Percentage of negative cells (e.g. no PI3K or MAPK pathway activation)
- Percentage of ERK1/2 activated cells (MAPK pathway activation)
- Percentage of Akt activated cells (PI3K pathway activation)
- Percentage of dual pathway activation (MAPK and PI3K pathway activation)
The Muse® PI3K/MAPK Dual Pathway Activation Kit includes two directly conjugated antibodies, a phospho-specific Akt (Ser473)-Alexa Fluor®555 and a phospho-specific ERK1/2 (Thr202/Tyr204, Thr185/Tyr187)-PECy5 conjugated antibody to assess the activation of both the PI3K and MAPK signaling pathways simultaneously in testing samples. This kit contains optimized fixation, permeabilization, and assay buffers to provide researchers with a complete solution for PI3K and MAPK cell signaling analysis.
