Violet Laser Reagents for Apoptosis
 | Apoptosis is morphologically and biochemically distinct from cell death by injury (necrosis). Since no single parameter fully defines cell death in all systems, it is often advantageous to use a multiparametric approach when studying apoptotic events and their temporal relationships. Two Molecular Probes® Vybrant® Apoptosis assay kits provide reagents to distinguish apoptotic cell populations from necrotic cell populations off the violet laser. Quick Links: |
Vybrant® Apoptosis Assay Kits
Apoptosis is a carefully regulated process of cell death that occurs as a normal part of development. It is distinguished from necrosis, or accidental cell death, by characteristic morphological and biochemical changes. Two Vybrant® Apoptosis Assay Kits incorporate a violet-excitable stain as part of a two-color assay for the detection of apoptosis. The Pacific Blue™–annexin conjugate detects changes in membrane asymmetry characteristic of apoptotic cells. In addition to changes in membrane asymmetry, slight changes in permeability to dyes such as PO-PRO™-1 (also available in Vybrant® Apoptosis Assay Kit #13 PO-PRO™-1/7-aminoactinomycin D) can be used to identify apoptotic cells. Unlike annexin conjugates, PO-PRO™-1 can provide efficient staining of both trypsinized and suspension cells.
- Detection of multiple apoptotic parameters on the violet laser
- Increased multiplexing capabilities
- Available as stand-alone reagents or easy-to-use kits
Violet-excited DNA stain for apoptosis detection. Jurkat cells were treated with 10 μM camptothecin for 4 hr and then stained with the reagents in the Vybrant® Apoptosis Assay Kit #13 PO-PRO™-1/7-aminoactinomycin D. Cells were analyzed by flow cytometry using 405 nm and 488 nm excitation. Apoptotic cells (A) are clearly distinguished from live cells (L) and dead cells (D).
| Product | |
|---|---|
| Annexin V, Pacific Blue™ Conjugate |  |
| Vybrant® Apoptosis Assay Kit #13 PO-PRO™-1/7-aminoactinomycin D | |
| Vybrant® Apoptosis Assay Kit #14 Annexin V-Pacific Blue™ | |
CountBright™ Absolute Counting Beads
CountBright™ absolute counting beads are a calibrated suspension of microspheres that are brightly fluorescent across a wide range of excitation and emission wavelengths (UV to 635 nm excitation and 385nm emission). CountBright™ absolute counting beads are mixed with the cell sample and assayed via flow cytometry. By comparing the ratio of bead events to cell events, absolute numbers of cells in the sample can be calculated. Because CountBright™ beads are mixed in the test sample, absolute cell counts using this single-platform method are more accurate and less complicated than cell concentration determined using multiple-platform testing. CountBright™ absolute counting beads can be used with any sample type, including no-wash/lysed whole blood.
Determination of live and dead cells and absolute cell number
Plot of calcein fluorescence collected with 530/30 nm bandpass filter vs. ethidium homodimer-1 fluorescence collected with 610/20 nm bandpass filter, showing clear separation of live and dead cells, as well as counting beads (C36950). A mixture of live and heat-killed Jurkat cells (human T-cell leukemia) was treated with the reagents in the LIVE/DEAD® Viability /Cytotoxicity Kit, counting beads were added, and the sample was analyzed by flow cytometry using 488 nm excitation.
Plot of calcein fluorescence collected with 530/30 nm bandpass filter vs. ethidium homodimer-1 fluorescence collected with 610/20 nm bandpass filter, showing clear separation of live and dead cells, as well as counting beads (C36950). A mixture of live and heat-killed Jurkat cells (human T-cell leukemia) was treated with the reagents in the LIVE/DEAD® Viability /Cytotoxicity Kit, counting beads were added, and the sample was analyzed by flow cytometry using 488 nm excitation.
| Product | |
|---|---|
| CountBright™ Absolute Counting Beads | |
Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit
The Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit is an assay for use on a violet laser flow cytometer that is an easy, efficient method for the detection of apoptosis. It works well on adherent and suspension cells and correlates with other indicators of apoptosis like caspase detection and changes in mitochondrial membrane potential.
It contains a violet ratiometric membrane asymmetry probe, F2N12S, which can monitor changes in membrane asymmetry that occur during apoptosis through a change in the relative intensity of the two emission bands of the dye. Unlike annexins, this assay does not need any special buffers or wash steps and is less affected by cell membrane damage commonly found during the physical or chemical removal steps when assaying adherent cells, therefore providing better data quality.
It contains a violet ratiometric membrane asymmetry probe, F2N12S, which can monitor changes in membrane asymmetry that occur during apoptosis through a change in the relative intensity of the two emission bands of the dye. Unlike annexins, this assay does not need any special buffers or wash steps and is less affected by 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
Violet Ratiometric Membrane Asymmetry Probe for apoptosis detection. Jurkat cells (T-cell leukemia, human) treated with 10 μM camptothecin for four hours (panels B and D) or untreated control (panels A and C). Cells were stained according to the protocol. 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. In panels A and B, living cells can be discriminated from apoptotic and dead cells by the relative intensities of the two emission bands from F2N12S. 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.
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