Apoptotic Plasma Membrane Assays for Flow Cytometry

Some of the earliest detectable apoptotic events involve the plasma membrane, including changes in membrane asymmetry and permeability. In addition to annexin V conjugates, Life Technologies provides unique assays to measure membrane changes under conditions where annexin V binding is problematic, such as in adherent cells, and without using special buffers.

See All Plasma Membrane Assays

Annexin Conjugates

In apoptotic cells, phosphatidyl serine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thus exposing PS to the external cellular environment. Annexin V labeled with a fluorophore or biotin can identify apoptotic cells by binding to PS exposed on the outer leaflet of the membrane. The Alexa Fluor® series of dyes has proven to make brighter and more photostable bioconjugates than other organic dyes with similar spectral characteristics.

  • Conjugated to Molecular Probes® dyes for increased sensitivity
  • Conjugates for all available lasers for increased multiplexing capabilities
  • Available as stand-alone reagents or easy-to-use kits

 

 

  


Figure 1. Jurkat cells (T-cell leukemia, human) treated with 10 μM camptothecin for four hours (right panel) or untreated (as control, left panel). Cells were  treated with Annexin V-Alexa Fluor® 488 to identify apoptotic cells and propidium iodide to identify dead cells, followed by flow cytometric analysis. Note that the camptothecin-treated cells (bottom panel) have a higher percentage of apoptotic cells  than the basal level of apoptosis seen in the control cells (top panel). A = apoptotic cells, L = live cells, D = dead cells.

Conjugate Laser Ex/Em Cat. No.  
Alexa Fluor® 350UV346/442A23202product details
Pacific Blue™ dye405410/455A35122product details
Alexa Fluor® 488488495/519A13201product details
R-PE488496, 546, 565/578A35111product details
Alexa Fluor® 555532555/565A35108product details
Alexa Fluor® 568561578/603A13202product details
Alexa Fluor® 647633/5650/660A23204product details

Violet Asymmetry Probe

The Violet Ratiometric Membrane Asymmetry Probe⁄Dead Cell Apoptosis Kit provides an easy, efficient method for the detection of apoptosis with dead cell discrimination using a violet laser flow cytometer. The Violet Ratiometric Membrane Asymmetry Probe is a novel violet excitable dye for the detection of membrane asymmetry changes during apoptosis. It works well on adherent and suspension cells and correlates with other indicators of apoptosis, such as caspase detection and changes in mitochondrial membrane potential.

The dye exhibits an excited-state intramolecular proton transfer (ESIPT) reaction resulting in a dual fluorescence with two emission bands corresponding to 530 nm and 585 nm, producing a two-color ratiometric response to variations in surface charge. The F2N12S probe is combined with SYTOX(R) AADvanced dead cell stain, which is capable of passing through the cell membrane only in late apoptotic or necrotic cells allowing discrimination form early apoptotic cells.

Unlike annexin-based assays, this assay does not require special buffers or wash steps, and it is less susceptible to the cell membrane damage commonly found during the physical or chemical removal steps when assaying adherent cells, therefore providing better data quality.

  • Accurate apoptotic analysis on trypsinized cells
  • Simple 5 minute staining protocol
  • Compatible with other blue-excited apoptotic stains

 

  


Figure 2. Violet Ratiometric Membrane Asymmetry Probe for apoptosis detection. Jurkat cells (T-cell leukemia, human) were treated with 10 μM camptothecin for four hours (panels B and D) or left untreated as a control (panels A and C). Samples were analyzed on a flow cytometer with 405 nm excitation using 585 nm and 530 nm bandpass filters for F2N12S, and 488 nm excitation for SYTOX® AADvanced™ dead cell stain using a 695 nm bandpass filter. Living cells can be discriminated from apoptotic and dead cells by the relative intensities of the two emission bands from F2N12S (A and B).  In panels C and D, SYTOX® AADvanced™ dead cell stain fluorescence is plotted against a derived ratio parameter from the two emission bands (585/530 nm) of F2N12S. A = apoptotic cells, L = live cells, D = dead cells.

Product Laser Ex/Em Apoptotic Cell Stain Dead Cell Stain Cat. No.  
Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit405405/585 (live), 405/530 (apoptotic), 546/647 (dead)F2N12SSYTOX® AADvanced™A35137product detail

Monomeric Cyanine Dyes

There are some situations where staining cells with annexin V is not the optimal method for detecting apoptosis. These include assays where cells are sensitive to the high calcium concentrations required for annexin V binding, assays where phosphatidylserine detection on adherent cells is adversely affected by trypsinization, and assays where washing of samples is prohibitive.

Three monomeric cyanine dyes (PO-PRO™-1, YO-PRO®-1, and TO-PRO®-3) have been shown to penetrate apoptotic cells because of permeability changes associated with the loss in asymmetry of the plasma membrane. These dyes enter apoptotic cells and bind to nucleic acids, while cell-impermeant dead cell stains are excluded. The three dyes have unique excitation wavelengths, providing enhanced flexibility in multiplexed assays.

 Figure 3. Detection of apoptosis using monomeric cyanine dyes. Induction of apoptosis was initiated in Jurkat cells with 10 µM camptothecin. The cells were then treated with YO-PRO®-1 dye (yellow) to detect apoptotic cells or propidium iodide to identify necrotic cells by flow cytometric analysis.

 

Product Laser Ex/Em Cat. No.  
PO-PRO™-1 iodide405435/455P3581product detail
YO-PRO®-1 iodide488491/509Y3603product detail
TO-PRO®-3 iodide633/5642/661T3605product detail