A Ratiometric Approach to Apoptosis Detection by Flow Cytometry

The Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit provides a simple and fast method for the detection of apoptosis with dead cell discrimination by flow cytometry. Suspension and adherent cells can be analyzed after a 5-minute incubation at room temperature without washing. The kit can be paired with other reagents such as the MitoProbe™ DiIC1(5) Assay Kit (mitochondria apoptosis assays), annexin V conjugates, or many others for multiparametric analysis of the apoptotic process. The kit can also be used alone as an endpoint apoptosis assay.


The Violet Ratiometric Membrane Asymmetry Probe (4'-N,N-diethylamino-6-(N-dodecyl-N-methyl-N-(3-sulfopropyl))ammoniomethyl-3-hydroxyflavone, F2N12S) is a novel dye for the detection of membrane asymmetry loss, which occurs during apoptosis [1]. The F2N12S dye exhibits an excited-state intramolecular proton transfer reaction resulting in dual fluorescence, with two emission bands corresponding to 530 nm and 585 nm using violet 405 nm excitation. The emitted fluorescence at both wavelengths is proportional to the change in membrane surface charge that occurs during apoptosis. The two distinct fluorescence emission spectra are also directly proportional to each other, a property that defines the dye as a ratiometric probe. The kit also includes SYTOX® AADvanced™ Dead Cell Stain (488 nm excitation, 647 nm emission) for discrimination of dead from live cells.

Advantages of a Ratiometric Probe

Ratiometric probes have several advantages over traditional fluorophore-labeled reagents. A ratiometric probe is self-referencing, because the ratio of emission at the two wavelengths is an absolute parameter that is independent of probe concentration, cell size, and instrument variation. Traditional fluorophore-labeled reagents, such as annexin V, often require titration to optimize the fluorescence separation between the live and apoptotic populations due to differences in cell size, concentration, and fluorophore. Optimizing a reagent may not be feasible if the samples are rare or difficult to produce. The dual-color fluorescence emission of the F2N12S reagent coupled with a derived ratio parameter enables researchers to resolve apoptotic and live cell populations among samples with cell concentrations that differ by as much as 100-fold, without the need to optimize the F2N12S probe concentration (Figure 1).


Figure 1. Resolution of live and apoptotic cell populations across a 100-fold difference in cell concentration by the Violet Ratiometric Asymmetry Probe/Dead Cell Apoptosis Kit. Flow cytometry density plots show control (Top Row) and camptothecin-treated (Middle Row) Jurkat cells (1 x 105 to 1 x 107 cells/mL) stained with 200 nM F2N12S and 1 μM SYTOX® AADvanced™ Dead Cell Stain (color in the plots simply reflects number of cells). Unlike some traditional fluorescent probes, the F2N12S ratio is not subject to probe depletion at high cell concentrations. (Bottom Row) As expected when cell concentration increases and probes become limiting, the fluorescence intensity of both emission bands (530 nm and 585 nm) of F2N12S decreases, but the ratio of fluorescence of the two bands only begins to be affected around 1 x 107 cells/mL. Even at 1 x 107 cells/mL, the apoptotic (A), live (L), and dead (D) cell populations are still clearly discernible, providing a robust assay for apoptosis when cell concentration cannot be accurately determined.

Apoptosis Data In As Little As 5 Minutes

The Violet Ratiometric Membrane Asymmetry Probe (F2N12S) and SYTOX® AADvanced™ Dead Cell Stain rapidly target the plasma membrane and nucleus. Live, apoptotic, and dead cell populations can be clearly resolved by flow cytometry, with F2N12S staining times as short as 30 seconds and optimal population resolution obtained after 5 minutes (Figure 2). The assay does not require special buffers, wash steps, or long incubation times; therefore, the chance of cells completing the apoptotic cycle and staining as dead, which can occur during extended sample processing, is reduced.


Figure 2. Optimal population resolution in 5 minutes using the Violet Ratiometric Asymmetry Probe/Dead Cell Apoptosis Kit. Control (Top Row) or camptothecin-treated (Bottom Row) Jurkat cells were incubated with 200 nM F2N12S for 30 seconds up to 30 minutes before analysis by flow cytometry. Samples were stained with 1 μM SYTOX® AADvanced™ Dead Cell Stain 5 minutes prior to addition of F2N12S to discriminate dead cells (D) from the apoptotic population (A). Populations of live (L) and apoptotic cells can be resolved in as little as 30 seconds after addition of F2N12S; this rapid analysis means fewer cells complete the apoptotic cycle during sample processing.

Enabling Multicolor Apoptosis Assays

The Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit can easily be paired with our other apoptosis reagents, enabling the development of diverse multiparameter apoptosis assays that are uniquely suited for the biological system of interest (Figure 3). The F2N12S dye, which is excited by 405 nm violet excitation, provides more flexibility to develop multiparameter, multiple-laser assays that utilize 405 nm, 488 nm, and 633 nm lasers, while reducing the need for complex compensation controls, because the F2N12S reagent has negligible absorbance at 488 nm and 633 nm. The Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit offers a rapid, sensitive, and fully multiplexable solution to expand the capability of today’s violet laser–equipped flow cytometers while providing a robust apoptosis assay for both suspension and adherent cell types.
Multiparametric Apoptosis Assay

Figure 3. Multiparameter apoptosis assay. Control (Top Row) and 4-hour camptothecin-treated (Bottom Row) Jurkat cells were stained with 1 μM SYTOX® AADvanced™ Dead Cell Stain, 200 nM F2N12S, and 50 nM MitoProbe™ DiIC1(5). Dead cells were first excluded (histograms) by gating live cells (those that have lower SYTOX® AADvanced™ fluorescence, indicated by bars). Bivariant density plots show a two-parameter apoptosis assay for mitochondrial membrane potential loss (decreased DiIC1(5) fluorescence) and breakdown of membrane asymmetry (smaller F2N12S 585/530 nm fluorescence ratio). The A, L, and D labels on the graphs indicate apoptotic, live, and dead cells, respectively.

References

  1. Shynkar VV, Klymchenko AS, Kunzelmann C et al. (2007) J Am Chem Soc 129:2187–2193.
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