Muse® Assays for Apoptosis Analysis

The degree of apoptosis in a cell population is an important parameter that contributes to a comprehensive picture of cell health. The assessment of cellular apoptosis has been limited due to the requirements for expensive and complicated instrument platforms, expertise and improved analytical methods that provide rapid, robust and reproducible apoptosis data. The Muse® Cell Analyzer delivers access to these improvements, facilitating apoptosis monitoring and thereby enabling the effcient, daily execution of cellular research.

Muse® apoptosis kits allow the study of early, mid, and late apoptosis, and also a pan-caspase assay for broad apoptosis activity:

  • Muse® Annexin V and Dead Cell (mid-late apoptosis)
  • Muse® Mitopotential (early apoptosis)
  • Muse® Caspase-3/7 (late apoptosis)
  • Muse® MultiCaspase (multiple apoptosis indicators)

Featured Kits

Muse® MitoPotential Assay (Cat. No. MCH100110)

Mitochondrial membrane potential changes have been implicated in apoptosis, necrotic cell death and caspase-independent cell death. Depolarization of the inner mitochondrial membrane potential is a reliable indicator of mitochondrial dysfunction and cellular health. This assay provides early and sensitive detection of cell health perturbation, enabling detection of mitochondrial depolarization under multiple treatment conditions in multiple cell types. The Muse® MitoPotential Assay uses the MitoPotential Reagent, a cationic, lipophilic dye, to detect changes the mitochondrial membrane potential, and 7-AAD as an indicator of cell death.

Four populations of cells can be distinguished in the assay:

  • Live cells with intact mitochondrial membrane: MitoPotential(+) and 7-AAD(-)
  • Live cells with depolarized mitochondrial membrane: MitoPotential(-) and 7-AAD(-)
  • Dead cells with depolarized mitochondrial membrane: MitoPotential(-) and 7-AAD(+)
  • Dead cells with intact mitochondrial membrane: MitoPotential(+) and 7-AAD(+)

Impact of apoptosis-inducing compounds on Jurkat cells (suspension line) and HeLa cells (adherent line) using the Muse® MitoPotential Assay. Dot plots show untreated cells (A and B) and cells treated with CCCP (C) and Staurosporine (D).

Muse® Annexin V & Dead Cell Assay (Cat. No. MCH100105)

This versatile assay can be used to assess health of both adherent and suspension cells under multiple treatment conditions and generate dose-response data on cells treated with apoptosis inducers. The assay is based on the binding of Annexin V to phosphatidylserine (PS) on the surface of apoptotic cells. It uses a premixed reagent containing fluorescently labeled Annexin V and a dead cell marker (7-AAD). Early in the apoptotic pathway, molecules of PS are translocated to the outer surface of the cell membrane where Annexin V can readily bind to them with high affinity. Late-stage apoptotic cells show loss of membrane integrity and uptake of membrane-impermeant 7-AAD.

The assay can thus distinguish four populations:

  • Non-apoptotic cells: Annexin V (-) and 7-AAD (-)
  • Early apoptotic cells: Annexin V (+) and 7-AAD (-)
  • Late stage apoptotic and dead cells: Annexin V (+) and 7-AAD (+)
  • Mostly nuclear debris: Annexin V (-) and 7-AAD (+)

Data output from the Muse® Annexin V and Dead Cell Assay. Jurkat cells were treated with the apoptosis inducer gambogic acid and analyzed with the Muse® Annexin V and Dead Cell Assay. Data output include summary statistics for the four populations (shown here) as well as optional dotplots (not shown).

Response of Jurkat cells treated with increasing concentrations of gambogic acid (apoptosis inducer) and analyzed using the Muse® Annexin V and Dead Cell Assay.

Muse® Caspase-3/7 Assay (Cat. No. MCH100108)

Caspase-3 and caspase-7 are "executioner caspases" that are activated downstream in the apoptosis cascade by a sequence of intrinsic or extrinsic signals. Once activated, these enzymes cause degradation of many key cellular proteins and influence chromatin condensation and DNA damage during apoptosis. Activation of caspase-3/7 is thus a hallmark and confrmation of the apoptotic process.

The Muse® Caspase-3/7 Assay determines the count and percentage of cells in various stages of apoptosis based on caspase 3/7 activity in combination with a dead cell dye.

The kit includes:

  1. The novel, fluorogenic Muse® Caspase-3/7 reagent for detecting caspase-3/7 activity
  2. Cell death dye, 7-AAD, that provides information on membrane integrity

The cell membrane-permeable Muse® Caspase-3/7 reagent contains a DNA-binding dye that is linked to a DEVD peptide substrate. While still conjugated to DEVD, the dye is unable to bind DNA. Cleavage by active caspase-3/7 in the cell results in release of the dye, translocation to the nucleus, binding of the dye to DNA and high fluorescence. The dead cell marker, 7-AAD, is excluded from live (healthy) and early apoptotic cells, but enters membrane-compromised, later-stage apoptotic and dead cells.

Four populations of cells can be distinguished in the assay:

  • Live cells: caspase-3/7(-) and 7-AAD(-)
  • Mid-apoptotic cells exhibiting caspase-3/7 activity: caspase 3/7(+) and 7-AAD(-)
  • Late apoptotic/dead cells: caspase-3/7(+) and 7-AAD(+)
  • Dead cells: caspase-3/7(-) and 7-AAD(+)

Impact of apoptosis-inducing compounds on HeLa cells and Jurkat cells analyzed using the Muse® Caspase-3/7 Assay.

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.

Muse® MultiCaspase Assay (Cat. No. MCH100109)

This assay detects the activity of caspases 1, 3, 4, 5, 6, 7, 8 and 9, which have multiple roles, in addition to carrying out apoptosis. The assay simultaneously determines the percentage and concentration of cells with caspase activity, in combination with a dead cell dye.

The kit includes:

  1. A fluorogenic, derivatized VAD-peptide that can detect the activity of multiple caspases
  2. A cell membrane impermeant dye, 7-AAD that provides information on cell membrane integrity

The VAD-peptide is derivatized with a fluorescent group and a fluoromethylketone irreversible caspase inhibitor moiety, generating a Fluorescent-Labeled Inhibitor of Caspases (FLICA). The peptide is membrane-permeable and non-cytotoxic. It binds to activated caspases with resulting fluorescent signal proportional to the number of active caspases in the cell. The dead cell marker, 7-AAD, is excluded from live (healthy) and caspase positive cells, but stains membrane-compromised, later-stage apoptotic and dead cells that show increased fluorescence in the viability axis.

Four populations of cells can be distinguished in the assay:

  • Live cells: caspase (-) and 7-AAD(-)
  • Cells exhibiting pan caspase activity: caspase(+) and 7-AAD(-)
  • Late caspase active/dead cells: caspase(+) and 7-AAD(+)
  • Dead cells: caspase(-) and 7-AAD(+)

Data output for the Muse® MultiCaspase Assay. Jurkat cells were treated with staurosporine, stained with the Muse® MultiCaspase Kit and analyzed on the Muse® Cell Analyzer. The summary data with statistics (top) show the percentages and the concentration (cells/mL) for the gated events in each quadrant, as well as the percentage and concentration of total caspase-positive cells. Data is also displayed as dot plots (bottom). The frst dotplot (left) shows cell size index vs. Caspase activity and the second plot (right) shows viability vs. caspase activity.

     
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