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VIABILITY AND APOPTOSIS ASSAYS FOR FLOW CYTOMETRY

Biotium offers a wide selection of assay kits for cell viability, cell death and apoptosis for flow cytometry. Jump to a section below to learn more:

Bright, fixable dead cell stains, available in 8 colors
Monitor apoptosis with the worlds only live-cell, real-time caspase-3 substrates
Available with a wide selection of CF® dyes and other labels, including Near-IR CF® Dye Annexin V conjugates
Kits containing combinations of dyes to measure cell viability, death and apoptosis
Dyes for monitoring mitochondrial membrane potential
Non-toxic, fixable proliferation dyes designed as improvements to CFSE

Knowledge Center

To learn more about the different apoptosis pathways and the ways to detect and measure apoptosis in your cells, download this eBook from The Scientist and Biotium titled “Visualizing Apoptosis as it Happens”.

 

 


LIVE-OR-DYE™ FIXABLE VIABILITY STAINING KITS

Live-or-Dye™ Fixable Viability Staining Kits

Live-or-Dye™ fixable dead cell stains are cell membrane impermeable amine-reactive dyes. The dyes enter dead cells that have compromised membrane integrity and covalently label free amines on intracellular proteins (Fig. 1).

Live-or-Dye™ labeling is extremely stable, allowing the cells to be fixed and permeabilized without loss of fluorescence or dye transfer between cells. The staining protocol has been optimized to maximize live/dead discrimination with minimal live cell staining, in order to prevent interference with immunostaining. Biotium offers a selection of eight bright and photostable Live-or-Dye™ stains for maximal flexibility in multi-color analysis (Fig. 2). We also offer Live-or-Dye NucFix™ Red, a unique fixable nuclear-specific dead cell stain.

Figure 1. Principle of Live-or-Dye™ discrimination of live and dead cells.

8 color options

Figure 2. Live-or-Dye™ Fixable Viability Staining Kits come in 8 fluorescent colors spanning the spectrum from UV to near-infrared. Live (open peaks) or heat-killed (solid peaks) Jurkat cells stained with Live-or-Dye™ Fixable Viability Stains and analyzed by flow cytometry. Results are comparable for live or fixed/permeabilized cells.

To learn more about Live-or-Dye™ Fixable Viability Stains, please visit the Live-or-Dye™ technology page.

Live-or-Dye™ Fixable Viability Staining Kits

Catalog No.

Viability Dye

Laser line

Emission filter

Abs/Em Max

Applications

32002, 32002-TLive-or-Dye™ 350/448355 nmDAPI or Violet347/448 nmFlow Cytometry
32003, 32003-TLive-or-Dye™ 405/452405 nmPacific Blue®408/452 nmFlow Cytometry
32009, 32009-TLive-or-Dye™ 405/545405 nmAmCyan395/545 nmFlow Cytometry,
Microscopy
32004, 32004-TLive-or-Dye™ 488/515488 nmFITC490/515 nmFlow Cytometry,
Microscopy
32012, 32012-TLive-or-Dye™ 510/550488 nmSpectral scan516/549 nmSpectral Cytometry
32005, 32005-TLive-or-Dye™ 568/583488 or 561 nmPE562/583 nmFlow Cytometry,
Microscopy
32006, 32006-TLive-or-Dye™ 594/614488 or 561 nmPE-Texas Red®593/614 nmFlow Cytometry,
Microscopy
32007, 32007-TLive-or-Dye™ 640/662633 or 640 nmAPC642/662 nmFlow Cytometry
32013, 32013-TLive-or-Dye™ 665/685633 or 640 nmSpectral scan667/685 nmSpectral Cytometry
32008, 32008-TLive-or-Dye™ 750/777633 or 640 nmAPC-Cy®7755/777 nmFlow Cytometry,
Microscopy
32010, 32010-TLive-or-Dye NucFix™ Red488 or 532 nmPE-Texas Red®520/593 nmFlow Cytometry,
Microscopy

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NUCVIEW® CASPASE-3 SUBSTRATES

NucView® Caspase-3 Substrates for live cell apoptosis monitoring

NucView® Caspase-3 Substrates are novel fluorogenic substrates developed by Biotium to detect apoptosis in intact cells in real-time. Biotium has invented a new concept in the design of fluorogenic enzyme substrates. The enzyme substrate is attached to a fluorogenic DNA dye. Before cleavage, the dye is non-fluorescent and unable to bind to DNA. Upon enzymatic cleavage of the substrate, the dye is released and becomes capable of binding to DNA to emit fluorescence. In the case of NucView® Caspase-3 Substrates, the caspase-3/7 substrate peptide DEVD is attached to a DNA-binding dye. The substrate enters the cell cytoplasm where it is cleaved by caspase-3 in apoptotic cells to release the fluorogenic DNA dye, which stains the nucleus. NucView® can be fixed with formaldehyde after staining, but it cannot be used to stain fixed cells or tissues.

Features

  • Simple, homogenous assay for endpoint assay or real-time detection in live cells
  • Non-toxic, allowing for multi-day experiments
  • Detect caspase-3 activity and visualize apopototic nuclear morphology
  • For flow cytometry, fluorescence microscopy, or live cell imaging systems
  • Available as stand-alone substrates or in kits with other apoptosis or necrosis probes
  • Available with blue, green, or red fluorescence
  • NucView® 488 validated in more than 100 cell types and 200 publications – See the list

Figure 5. Principle of NucView® substrate technology. The substrate is non-fluorescent and does not bind DNA. After enzyme cleavage, the high-affinity DNA dye is released to bind DNA and become fluorescent.
Figure 6. NucView® 405 Caspase-3 Substrate fluorescence over time in staurosporine-treated Jurkat cells, analyzed in the Pacific Blue® channel of a BD LSRII flow cytometer.

To learn more about NucView® Caspase-3 substrates, and kits please visit the NucView® technology page.

NucView® Caspase-3 Substrates and Kits

Catalog No.

Features

NucView® 405 Caspase-3 Substrate, 1 mM in DMSO10405Blue fluorescence for flow cytometry in the Pacific Blue® channel or microscopy with the 405 nm laser
NucView® 405 Caspase-3 Substrate, 1 mM in PBS10407NucView® 405 substrate in PBS, for DMSO-sensitive cell types
NucView® 488 Caspase-3 Substrate, 1 mM in DMSO10402Green fluorescent substrate validated in more than 100 cell types and 200 publications
NucView® 488 Caspase-3 Substrate, 1 mM in PBS10403NucView® 488 substrate in PBS, for DMSO-sensitive cell types
NucView® 530 Caspase-3 Substrate, 1 mM in DMSO10406Orange fluorescence for microscopy in the Cy®3 channel or flow cytometry in the R-PE channel
NucView® 530 Caspase-3 Substrate, 1 mM in PBS10408NucView® 530 substrate in PBS, for DMSO-sensitive cell types
NucView® 488 and MitoView™ 633 Apotosis Detection Kit30062NucView® 488 and far-red fluorescent MitoView™ 633 for the Cy®5 channel
NucView® 488 and RedDot™ 2 Apoptosis & Necrosis Kit30072NucView® 488 and far-red dead cell DNA dye RedDot™ 2 for the Cy®5 channel
Dual Apoptosis Assay with NucView® 488 and CF®594 Annexin V30067NucView® 488 and red fluorescent Annexin V apoptosis probe
Dual Apoptosis Assay with NucView® 488 and CF®640R Annexin V30073NucView® 488 and far-red fluorescent Annexin V apoptosis probe

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ANNEXIN V CONJUGATES

Annexin V is a 36 kDa protein that has high affinity for phosphatidylserine (PS). During apoptosis, PS is translocated from the inner to the outer leaflet of the plasma membrane, where it can be stained by fluorescent conjugates of Annexin V. Annexin V conjugates are available with a wide selection of CF® dyes and other labels, including Near-IR CF® Dye Annexin V conjugates.

We also offer Annexin V conjugates in an azide-free formulation, for long-term or in vivo studies.


Jurkat cells were treated with staurosporine to induce apoptosis (pink), or with DMSO as a negative control (blue) for the times indicated, then stained for 15 minutes at room temperature with NucView® 530 Caspase-3 Substrate (x-axis) and CF®640R Annexin V (y-axis).

ProductEx/EmConcentrationSizeCatalog No. / Purchase
Biotin Annexin VN/A50 ug/mL0.5 mLPurchase 29013
CF®350 Annexin V 347/448 nm50 ug/mL0.5 mLPurchase 29012
CF®405M Annexin V 408/452 nm50 ug/mL0.5 mLPurchase 29009
CF®450 Annexin V 405/460 nm50 ug/mL0.5 mLPurchase 29083
CF®488A Annexin V 490/515 nm50 ug/mL0.5 mLPurchase 29005
CF®555 Annexin V 555/565 nm50 ug/mL0.5 mLPurchase 29004
CF®568 Annexin V 562/583 nm50 ug/mL0.5 mLPurchase 29010
CF®594 Annexin V 593/614 nm50 ug/mL0.5 mLPurchase 29011
CF®633 Annexin V 630/650 nm50 ug/mL0.5 mLPurchase 29008
CF®640R Annexin V 642/662 nm50 ug/mL0.5 mLPurchase 29014
CF®647 Annexin V 650/665 nm50 ug/mL0.5 mLPurchase 29003
CF®660R Annexin V 663/682 nm50 ug/mL0.5 mLPurchase 29069
R-PE Annexin V 496, 546, 565/578 nm5 uL/test100 uLPurchase 29045-100uL
500 uLPurchase 29045-500uL
APC Annexin V 650/660 nm5 uL/test100 uLPurchase 29057-100uL
500 uLPurchase 29057-500uL
FITC Annexin V 496/516 nm50 ug/mL0.5 mLPurchase 29001
Sulforhodamine 101 (Texas Red®) Annexin V 591/608 nm50 ug/mL0.5 mLPurchase 29002
5X Annexin V Binding BufferN/A5X15 mLPurchase 99902

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FLUORESCENT VIABILITY STAINING KITS

Viability/Cytotoxicity Assay Kit

The Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells stains live cells with green fluorescence and dead cells with red fluorescence for detection by fluorescence microscopy or flow cytometry. Calcein-AM is a non-fluorescent cell-permeable ester that can passively enter cells. In viable cells, it is hydrolyzed by esterases in the cytoplasm to release the green fluorescent dye calcein. Calcein itself is membrane-impermeant and is only retained in viable cells with intact plasma membranes. Ethidium Homodimer III is a red fluorescent membrane-impermeant nucleic acid dye. It selectively stains dead cells with damaged plasma membranes (necrotic or late apoptotic cells).

Apoptosis/Necrosis Kits

We offer many different staining kits with various combinations of dyes, including: NucView® caspase-3 substrate, our bright and photostable CF®488A Annexin V, a variety of dead cell nucleic acid stains, including propidium iodide (PI), 7-AAD, Ethidium Homodimer III (EthDIII), and RedDot™2.


HeLa cells stained with the Viability/Cytotoxicity Assay for Animal Live & Dead Cells. Dead cells are stained red and live cells are stained green.

ProductCatalog No.Features
CF®488A Annexin V and PI Apoptosis Kit30061• CF®488A Annexin V stains apoptotic cells green for the FITC channel
• PI stains necrotic cells red for the PE channel
CF®488A Annexin V and 7-AAD Apoptosis Kit30060• CF®488A Annexin V stains apoptotic cells green for the FITC channel
• 7-AAD stains necrotic cells red for the PE-Cy®5 channel
Apoptosis & Necrosis Quantitation Kit Plus30065• CF®488A Annexin V stains apoptotic cells green for the FITC channel
• EthDIII stains dead cells red for the PE channel
Apoptotic, Necrotic & Healthy Cells Quantitation Kit Plus30066• CF®488A Annexin V stains apoptotic cells green for the FITC channel
• EthDIII stains dead cells red for the PE channel
• Hoechst stains all cell nuclei blue for the Pacific Blue channel
NucView®488 and RedDot™2 Apoptosis and Necrosis Kit30072• NucView®488 stains apoptotic nuclei green for the FITC channel
• RedDot™2 stains necrotic cells far-red for the APC channel
Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells30002• Green dye Calcein AM detects intracellular esterase activity in live cells
• EthDIII stains dead cells red for the PE channel

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MITOCHONDRIAL MEMBRANE POTENTIAL DYES

Loss of mitochondrial membrane potential is a hallmark for apoptosis. Biotium offers MitoView™ 633 dye for membrane potential-sensitive staining of mitochondria by microscopy or flow cytometry. For simultaneous detection of mitochondrial membrane potential and caspase-3 activity, see the NucView®488 and MitoView™633 Apoptosis Assay.

We also offer MitoView™ 405, which changes localization upon mitochondrial depolarization, and MitoView™ Green, a membrane-potential independent mitochondrial dye that can be used to image mitochondria following mitochondrial depolarization, or after fixation.

In healthy cells, JC-1 dye aggregates in mitochondria as a function of membrane potential, resulting in red fluorescence with brightness proportional to the membrane potential. Conversely, in apoptotic and necrotic cells with diminished mitochondrial membrane potential, JC-1 exists in a green fluorescent monomeric form in the cytosol, allowing of cell viability to be assessed by measuring the ratio of red to green fluorescence by flow cytometry or fluorescence microplate reader.Rhodamine 123 is a green fluorescent mitochondrial membrane potential dye. Red fluorescent TMRM and TMRE are the preferred dyes for quantitative membrane potential measurements.


 

Timecourse of NucView®488 and MitoView™633 in staurosporine-treated Jurkat cells. As apoptosis progresses, MitoView™633 staining is lost and cells become positive for NucView®488.

Mitochondrial Dyes

Abs/Em

Detection channel

Potential-dependent?

Catalog no.

Size

MitoView™ 633622/648 nm*Cy®5, APC*Yes70055-T50 ug
7005520×50 ug
NucView® 488 & MitoView™ 633 Apoptosis Kit500/530 nm (NucView®488)
622/648 nm* (MitoView­™ 633)
FITC/Cy®5*Yes30062100 assays
MitoView™ 405398/440 nmDAPIPartial†70070-T50 ug
7007020×50 ug
MitoView™ Green490/523 nmFITC, GFPNo70054-T50 ug
7005420×50 ug
JC-1 Mitochondrial Membrane Potential Detection Kit510/527 nm (cytoplasm)
585/590 nm (mitochondria)
FITC/Cy®3Yes30001100 assays
JC-1 Chloride Salt700115 mg
JC-1 Iodide Salt700145 mg
Rhodamine 123505/534 nmFITCYes7001050 mg
TMRE548/573 nmCy®3Yes7001625 mg
TMRE, 2 mM in DMSO700160.5 mL
TMRM548/573 nmCy®3Yes7001725 mg

* MitoView™ 633 also has visible red fluorescence in the Cy®3/rhodamine channel. It is not recommended for imaging with other visible red probes.
† Localizes to the cytoplasm after mitochondrial depolarization, but still retains fluorescence.
Cy Dye is a registered trademark of GE Healthcare.

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VIAFLUOR® SE CELL PROLIFERATION KITS

Cell Proliferation Monitoring by Flow Cytometry

Cell proliferation dyes diffuse passively into live cells and are used for long-term cell labeling. They are initially non-fluorescent esters of amine-reactive dyes, but are converted to fluorescent dyes by intracellular esterases. The dyes then covalently react with amine groups on intracellular proteins, forming fluorescent conjugates that are retained in the cell. Immediately after staining a single, bright fluorescent population will be detected by flow cytometry. Each cell division that occurs after labeling is revealed by the appearance of a successively dimmer fluorescent peak on a flow cytometry histogram (Figs. 3&4). Cell proliferation dyes can be used to track cell divisions in vivo or in vitro. The staining can withstand fixation and permeabilization for subsequent immunostaining.

ViaFluor® SE Cell Proliferation Kits

CFSE (also known as CFDA-SE or carboxyfluorescein diacetate, succinimidyl ester) has been used for many years to monitor cell proliferation by flow cytometry. While several alternatives have been developed in more recent years, CFSE remains popular and widely used. Biotium continues to offer CFSE under the tradename ViaFluor® CFSE. However, CFSE has several drawbacks including leakage from the cell, cell toxicity, and bleed-through into the PE and PE-TexasRed® channels.

ViaFluor® 405 SE and ViaFluor® 488 SE cell proliferation dyes were developed at Biotium to provide superior cell staining, fixability, and low toxicity. ViaFluor® 405 is excited with the violet laser and detected in the Pacific Blue® channel, and gives great peaks with no toxicity (Fig. 3). It behaves identially to the popular proliferation dye CellTrace™ Violet (Fig. 3). ViaFluor® 488 and ViaFluor® CFSE are both detected in the FITC channel, but ViaFluor® 488 was developed as a less toxic, less leaky and more fixable alternative to the classic dye CFSE (Fig. 4). It also has less bleed-through into other channels such as PE and PE-Texas-Red®.

Figure 3. Jurkat cells were stained with 5 uM cell proliferation dye: ViaFluor® 405 (left panel) or CellTrace™ Violet (right panel). Cells were analyzed in the Pacific Blue channel immediately after staining, and then every day for four days. Both dyes give sharp peaks showing the progressive decrease in signal caused by cell division.
Figure 4. Jurkat cells were stained with 3 uM cell proliferation dye: ViaFluor® 488 (left panel) or ViaFluor® CFSE (right panel). Cells were analyzed in the FITC channel immediately after staining, and then every day for three days. CFSE loses a lot of signal between initial staining and Day 1, limiting the number of cell divisions that can be measured, while ViaFluor® 488 retains dye better and gives nice sharp peaks for each division cycle.

To learn more about ViaFluor® Cell Proliferation Kits, please visit the ViaFluor® technology page.

Cell Division Tracking Dyes

Catalog No.

Ex/Em (nm)

Flow detection channel

Features

ViaFluor® 405 Cell Proliferation Kit30068/30068-T408/452 Pacific Blue®
  • Track cell division by dye dilution using flow cytometry

  • ViaFluor® 488 is a unique, improved green dye to replace CFSE

  • ViaFluor® 405 works as well as CellTrace™ Violet
ViaFluor® 488 Cell Proliferation Kit30086/30086-T493/532FITC
ViaFluor® CFSE Cell Proliferation Kit30050495/519FITC