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CELL VIABILITY AND APOPTOSIS

Biotium offers a wide-selection of assay kits for cell viability and cell death for microplate reader, flow cytometry, or fluorescence microscopy. Jump to a section below to learn more:


LIVE CELL QUANTITATION ASSAYS FOR MICROPLATE READER


Absorbance-based cell quantitation assays

MTT, XTT, and resazurin are reduced by active mitochondria to yield colored products, and thus are useful for assaying cell viability and quantitating cell number. MTT and XTT are reduced to colored formazin salts that can be measured by absorbance. Resazurin is reduced to form resorufin, which can be measured by either absorbance or fluorescence. These are easy-to-use homogeneous assays, but they have the disadvantage of requiring several hours for development. In addition, drugs or compounds with redox activity, like antioxidants, can interfere with the assays by directly affecting substrate reduction. MTT assay requires cell lysis before the absorbance can be measured. XTT and resazurin do not require cell lysis, allowing measurements to be made at multiple timepoints.

Absorbance Assays

  • Quantitate cells based on their metabolic activity
  • Simple, homogeneous assays
  • Usually require several hours development time
  • Compounds like antioxidants can interfere with assays

Fluorescence-based cell quantitation assays

Fluorescence-based assays generally have higher sensitivity and faster assay times compared to absorbance-based assays. Resazurin (equivalent to alamarBlue® or CellTiter-Blue®) is a non-fluorescent blue dye that is reduced by mitochondrial metabolic activity to form pink fluorescent resorufin. It can be measured by fluorescence or absorbance, and does not require cell lysis, allowing multiple measurements to be made over time.

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, so it is a true endpoint assay for cell viability. Also see our Viability/Cytotoxicity Assay for Animal Live & Dead Cells, which combines calcein-AM with the fluorescent dead cell stain EthD-III, and is compatible with microplate reader.

Fluorescence Assays

  • Fluorescence-based assays for microplate reader
  • Use Resazurin to quantitate cells based on metabolic activity
  • Calcein-AM measures cellular enzymatic activity and membrane integrity
  • Highly sensitive, simple, and rapid

Viability Assays for Microplate Reader

Catalog No.

Detection

Features

MTT Cell Viability Assay Kit 30006 Absorbance at 570 nm • Substrates reduced by mitochondrial activity to form colored formazen salts
• Widely used, simple, homogeneous assays
• MTT requires cell lysis before detection, XTT does not
XTT Cell Viability Assay Kit 30007 Absorbance at 475 nm
Resazurin Cell Viability Assay Kit 30025 Absorbance at 570 nm or
Fluorescence at 571/585 nm
• Simple, homogeneous assay for mitochondrial metabolic activity
• Measure absorbance or fluorescence without cell lysis
• Equivalent to alamarBlue® or CellTiter-Blue® at lower cost
Calcein-AM Cell Viability Assay Kit 30026 Fluorescence at 485/530 nm • True endpoint assay for cell viability
• Measures cellular enzymatic activity and membrane integrity
• Simple, rapid assay

alamarBlue is a registered trademark of Trek Biosystems. CellTiter-Blue is a registered trademark of Promega Corporation.

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LUMINESCENCE-BASED CELL QUANTITATION

ATP-Glo™ Bioluminometric Cell Viability Assay

ATP-Glo™ is a highly sensitive and linear luminescence-based assay that allows quantitation of viable mammalian cells based on their ATP levels. The assay can detect as little as 0.01 pmol ATP or a single viable cell. It is a homogeneous assay for use with cells in culture medium. Note: ATP-Glo™ is a flash-type assay that requires samples to be read in a luminometer immediately after adding the reagent, or using a luminescence plate reader with a reagent injector.

Features

  • Economical luminescence-based cell or ATP quantitation
  • Highly sensitive and linear
  • Homogeneous assay, add reagent directly to cells in medium
  • Flash-type assay requires samples to be read immediately after adding reagent

ATP-Glo™ Kits

Catalog No.

Size

ATP-Glo™ Bioluminometric Cell Viability Assay 30020-T 50 assays
30020-1 200 assays
30020-2 1000 assays
Quantitation of an ATP standard curve (red squares), or a dilution series of live Jurkat cells (blue diamonds) with the ATP-Glo™ Assay Kit.

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CELL DIVISION TRACKING BY FLOW CYTOMETRY


ViaFluor® SE Cell Proliferation Kits

ViaFluor® SE Cell Proliferation Kits can be used to monitor cell division by flow cytometry. ViaFluor® SE dyes are membrane-permeant compounds that are initially non-fluorescent esters, but are converted to fluorescent dyes by intracellular esterases and will covalently react with amine groups on intracellular proteins at the same time, forming fluorescent conjugates that are retained in the cell. Immediately after staining, a single bright fluorescent population will be detected by flow cytometry. With each cell division, daughter cells inherit roughly half of the fluorescent label, allowing the number of cell divisions that occur after labeling to be detected by the appearance of successively dimmer fluorescent peaks on a flow cytometry histogram compared to cells analyzed immediately after staining. Cell proliferation dyes can be used to track cell divisions in vivo or in vitro. The staining also can withstand fixation and permeabilization for subsequent immunostaining. Available with blue, green, and red fluorescence.

Top: Principal of dye dilution assays for cell proliferation. Bottom: Cell division tracking in Jurkat cells over the course of 5 days (d0-d4). Cells were labeled with ViaFluor®405 on day 0, and analyzed by flow cytometry on each following day. Each successively dimmer peak represents one cell division. Unstained cells are in gray.

Cell Division
Tracking Dyes

Catalog No.

Ex/Em (nm)

Flow detection
channel

Features

ViaFluor® 405 Cell Proliferation Kit 30068 408/452 Pacific Blue® • Track cell division by dye dilution using flow cytometry

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

• ViaFluor® 405 replaces CellTrace™ Violet
ViaFluor® 488 Cell Proliferation Kit 30086 493/532 FITC
ViaFluor® CFSE Cell Proliferation Kit 30050 495/519 FITC

Pacific Blue and Texas Red are registered trademarks of Thermo Fisher Scientific.

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MITOCHONDRIAL MEMBRANE POTENTIAL DYES

MitoView™ 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.

JC-1 and Other Classic Mitochondrial Membrane Potential Dyes

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.

MitoView™ 405
MitoView™ Green
MitoView™ 633

Mitochondrial Dyes

Abs/Em

Detection channel

Potential-dependent?

Catalog No.

Size

MitoView™ 633 622/648 nm* Cy®5, APC* Yes 70055-T 50 ug
70055 20×50 ug
NucView® 488 & MitoView™ 633 Apoptosis Kit 500/530 nm (NucView®488)
622/648 nm* (MitoView­™ 633)
FITC/Cy®5* Yes 30062 100 assays
MitoView™ 405 398/440 nm DAPI Partial† 70070-T 50 ug
70070 20×50 ug
MitoView™ Green 490/523 nm FITC, GFP No 70054-T 50 ug
70054 20×50 ug
JC-1 Mitochondrial Membrane Potential Detection Kit 510/527 nm (cytoplasm)
585/590 nm (mitochondria)
FITC/Cy®3 Yes 30001 100 assays
JC-1 Chloride Salt 70011 5 mg
JC-1 Iodide Salt 70014 5 mg
Rhodamine 123 505/534 nm FITC Yes 70010 50 mg
TMRE 548/573 nm Cy®3 Yes 70016 25 mg
TMRE, 2 mM in DMSO 70005 0.5 mL
TMRM 548/573 nm Cy®3 Yes 70017 25 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 Cytiva.

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LIVE & DEAD CELL STAINING

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).

Cytotoxicity/Viability Assay

Catalog No.

Size

Viability/Cytotoxicity Assay for Animal Live & Dead Cells 30002-T 150 assays
30002 300 assays


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

Dead Cell Nucleic Acid Stains

Membrane-impermeant nucleic acid dyes can be used to selectively stain necrotic and late apoptotic cells that have leaky plasma membranes. These dyes have very low fluorescence until they bind DNA or RNA, so they can be used to stain cells without a wash step for homogenous and real-time imaging in cell culture medium. Biotium offers dead cell stains in a variety of colors, as well as Apoptosis & Necrosis Staining Kits with dead cell stains and apoptosis markers.

In general, dead cell nucleic acid dyes cannot be fixed after staining, because the dyes do not become cross-linked to DNA. After fixation, they are free to diffuse from dead cells to live cells (which become permeabilized during fixation), resulting in poor discrimination between live and dead cell populations. For fixable dead cell stains, see our Live-or-Dye™ product line, including the nuclear-specific Live-or-Dye NucFix™ Red.

Dead Cell Nucleic Acid Stains

Catalog No.

Color (Ex/Em*)

Features

Oxazole Blue, 1 mM in DMSO 40091 Blue
(434/457 nm)
• Blue cell-impermeant dye
• Selectively stains early apoptotic cells
• Equivalent to PO-PRO™-1 Iodide
Oxazole Blue Homodimer, 1 mM in DMSO 40093 Blue
(433/457 nm)
• Blue cell-impermeant dye
• Equivalent to POPO™-1 Iodide
NucSpot® 470, 1000X in DMSO 40083 Green
(460/546 nm)
• Green cell-impermeant dye
• Nuclear-specific counterstain in fixed cells
• Useful for cell cycle analysis in fixed cells
• Excellent match for blue LED excitation sources
Oxazole Yellow, 1 mM in DMSO 40089 Green
(491/506 nm)
• Green cell-impermeant dye
• Selectively stains early apoptotic cells
• Equivalent to YO-PRO®-1 Iodide
Oxazole Yellow Homodimer, 1 mM in DMSO 40090 Green
(491/508 nm)
• Green cell-impermeant dye
• Equivalent to YOYO®-1 Iodide
TO Iodide, 1 mM in DMSO 40088 Green
(515/531 nm)
• Green cell-impermeant dye
• Equivalent to TO-PRO®-1 Iodide
Thiazole Orange Homodimer,
1 mM in DMSO
40079 Green
(514/531 nm)
• High affinity dimeric cyanine dye
• Dead cell stain and electrophoresis dye
• Equivalent to TOTO®-1 Iodide
NucSpot® 555/570 Nuclear Stain 41033 Red
(559/566 nm)
• Red cell-impermeant dye for the Cy®3 or PE channels
• Nuclear-specific counterstain in fixed cells
NucSpot® 568/580 Nuclear Stain 41036 Red
(572/583 nm)
• Red cell-impermeant dye for the Cy®3 or PE channels
• Nuclear-specific counterstain in fixed cells
• Suitable for multi-day live cell imaging
NucSpot® 594/615 Nuclear Stain 41037 Red
(603/613 nm)
• Red cell-impermeant dye for the Texas Red® or PE-Texas Red® channels
• Nuclear-specific counterstain in fixed cells
• Suitable for multi-day live cell imaging
Propidium Iodide, 100 mg 40016 Red
(530/622 nm)
• Widely used dead cell stain
• Can be excited by 488 nm laser line for detection in the PE channel by flow cytometry
• Useful for cell cycle analysis in fixed cells (with RNase treatment)
Propidium Iodide, 1 mg/mL in Water 40017
Propidium Iodide Buffer, 50 ug/mL 40048
Ethidium Homodimer I, 1 mg 40010 Red
(527/624 nm)
• High-affinity membrane-impermeant nucleic acid stain
• >30-fold fluorescence enhancement upon binding to DNA/RNA
• High-purity grade not available from other manufacturers
Ethidium Homodimer I, 2 mM in DMSO 40014
Ethidium Homodimer III, 1 mg 40050 Red
(532/625 nm)
• Developed at Biotium as an alternative to Ethidium Homodimer I
• 45% brighter than EthDI when bound to DNA
Ethidium Homodimer III, 1 mM in DMSO 40051
Oxazole Red, 1 mM in DMSO 40105 Far-red (613/629 nm) • Far-red cell-impermeant dye for the PE-Cy®5, or APC channel
• Useful dead cell stain
• Equivalent to YO-PRO®-3
Oxazole Red Homodimer, 1 mM in DMSO 40106 Far-red (612/631 nm) • Far-red cell-impermeant dye for the PE-Cy®5, or APC channel
• Useful dead cell stain
• Equivalent to YOYO®-3
7-AAD, 1 mg 40037 Far-red
(546/647 nm)
• Far-red dye for flow cytometry detection in the PE-Cy®5 channel
• Can be excited by the 488 nm or 532 nm laser line
• Useful for cell cycle analysis in fixed cells
7-AAD, 1 mg/mL solution 40084
NucSpot® Far-Red, 1000X in DMSO 40085 Far-red
(597/667 nm)
• Designed as improved replacement for 7-AAD
• For flow cytometry in the PE-Cy®5 or APC channel
• Useful for cell cycle analysis in fixed cells
• Less bleed into the PE-Texas Red® channel
RedDot™2 Far-Red Nuclear Stain 40061 Far-red
(665/695 nm)
• Far-red cell-impermeant dye for the Cy®5 channel
• Nuclear-specific counterstain in fixed cells
• Replaces Draq7™
Thiazole Red, 1 mM in DMSO 40087 Far-red
(642/657 nm)
• Far-red cell-impermeant dye for the Cy®5 channel
• Dead cell stain and electrophoresis dye
• Equivalent to TO-PRO®-3 Iodide
Thiazole Red Homodimer,
1 mM in DMSO
40080 Far-red
(642/661 nm)
• High affinity dimeric cyanine dye for the Cy®5 channel
• Useful dead cell stain
• Equivalent to TOTO®-3 Iodide
NucSpot® 650/665 Nuclear Stain 41034 Far-red
(653/671 nm)
• Far-red cell-impermeant dye for the Cy®5 or APC channels
• Nuclear-specific counterstain in fixed cells
NucSpot® 680/700 Nuclear Stain 41035 Near-IR
(683/707 nm)
• Near-IR cell-impermeant dye for the Cy®5.5 channel
• Nuclear-specific counterstain in fixed cells
NucSpot® 750/780 Nuclear Stain 41038 Near-IR
(757/780 nm)
• Near-IR cell-impermeant dye for the Cy®7 or APC-Cy®7 channels
• Nuclear-specific counterstain in fixed cells
• Suitable for multi-day live cell imaging
* When bound to nucleic acids
SYBR, PO-PRO, POPO, Texas Red, TOTO, TO-PRO, YO-PRO, and YOYO are trademarks or registered trademarks of Thermo Fisher Scientific. Cy Dye is a registered trademark of Cytiva. Draq7 is a trademark of Biostatus Ltd.

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FIXABLE LIVE & DEAD STAINS

Live-or-Dye™ Fixable Dead Cell Stains

Live-or-Dye™ Fixable Viability Stains are designed for discrimination between live and dead cells. The dyes are cell membrane impermeable and amine-reactive. They enter dead cells with damaged plasma membranes and covalently label intracellular proteins. The dyes react with surface proteins on live cells, but because these are much less abundant than intracellular proteins the staining is very low. Live-or-Dye™ labeling is extremely stable, so cells can be fixed and permeabilized without loss of fluorescence or dye transfer between cells. Biotium offers a selection of 14 bright and photostable Live-or-Dye™ stains for maximal flexibility in multi-color analysis.


Principle of Live-or-Dye discrimination of live and dead cells.
Live-or-Dye™ kits are available with 14 bright and stable dye colors for multichannel flow cytometry.

Flow Cytometry

Live/dead stains are useful probes to include when analyzing cell surface protein expression by flow cytometry, because they allow intracellular fluorescence signal from dead cells with permeable plasma membranes to be excluded from analysis. In addition, several Live-or-Dye™ stains were designed specifically for spectral flow to fill in gaps between common fluorophores: Live-or-Dye™ 510/550, 665/685, 375/600, and 615/740.

Sampler Kits Available

Not sure which kit is right for you? Live-Or-Dye™ stains are also available in sampler kits that include 5 spectrally compatible stains for either common flow cytometry configurations or spectral cytometry. View purchasing information below for details.

Fluorescence Microscopy

The staining protocol has been optimized to maximize live/dead discrimination with minimal live cell staining, in order to prevent interference with immunostaining. In microscopy, live/dead stains allow unambiguous visual discrimination of dead cells.

Ethanol-treated HeLa cells stained with Live-or-Dye™ Fixable Dead Cell Stains. Dead cells stain brightly with Live-or-Dye™, while live cells show minimal staining. Nuclei are stained with Hoechst (blue).

Live-or-Dye NucFix™ Red

Live-or-Dye NucFix™ Red is a unique, cell membrane impermeable dye that specifically stains the nuclei of dead cells. Unlike other commonly used nuclear stains such as propidium iodide or DRAQ7™, Live-or-Dye NucFix™ labeling is covalent, so the dye doesn’t transfer between cells after fixation. Live-or-Dye NucFix™ Red can be used for flow cytometry or fluorescence microscopy.

live or dye nucfix red
Discrimination of live and dead cells using Live-or-Dye NucFix™ Red Fixable Viability Stain. A. Flow cytometry of live (open peak) or heat-killed (solid peak) Jurkat cells stained with Live-or-Dye NucFix™ Red. B. Ethanol-treated HeLa cells stained with NucFix™ Red. Killed cells show bright red nuclear staining, compared to no staining seen in live cells. Nuclei are stained with Hoechst (blue).

Cat. No.

Viability Dye

Compatible lasers (nm)

Optimal detection channels

Notes

32018, 32018-T Live-or-Dye™ 330/410 355, 375 BUV395
32002, 32002-T Live-or-Dye™ 350/448 355, 375 DAPI
32014,32014-T Live-or-Dye™ 375/600 355, 375, 405 Spectral scan, BUV615, BV605 Developed for spectral cytometry
32003, 32003-T Live-or-Dye™ 405/452 405 Pacific Blue®, BV421, BV450
32009, 32009-T Live-or-Dye™ 405/545 405 AmCyan, BV510
32004, 32004-T Live-or-Dye™ 488/515 488 FITC Validated for microscopy
32012, 32012-T Live-or-Dye™ 510/550 488, 532 Spectral scan Developed for spectral cytometry
32005, 32005-T Live-or-Dye™ 568/583 488, 532, 561 PE, PI Validated for microscopy
32006, 32006-T Live-or-Dye™ 594/614 488, 532, 561 PI, PE-CF®594, PE-Texas Red® Validated for microscopy
32015, 32015-T Live-or-Dye™ 615/740 633-640 Spectral scan, APC-Cy®7 Developed for spectral cytometry
32007, 32007-T Live-or-Dye™ 640/662 633-640 APC Validated for microscopy
32013, 32013-T Live-or-Dye™ 665/685 633-640 Spectral scan, AF700 Developed for spectral cytometry
32008, 32008-T Live-or-Dye™ 750/777 633-640, 785 APC-Cy®7 The brightest dye for the APC-Cy®7 channel
32011, 32011-T Live-or-Dye™ 787/808 785, 808 APC-Cy®7, IR800

Live-or-Dye™ Sampler Kits

Kit Name

Catalog No.

Included dyes: Ex/Em (Cat No.)

Application

Live-or-Dye™ Fixable Viability Sampler Kit, Standard 32016 • 350/448 (32002A)
• 405/545 (32009A)
• 488/515 (32004A)
• 568/583 (32005A)
• 640/662 (32007A)
Designed for use on most standard flow cytometry laser and filter configurations
Live-or-Dye™ Fixable Viability Sampler Kit, Spectral 32017 • 350/448 (32002A)
• 375/600 (32014A)
• 510/550 (32012A)
• 615/740 (32015A)
• 665/685 (32013A)
Designed for use in spectral flow cytometry, to fill in gaps between common fluorophores

Draq7 is a trademark of Biostatus, Ltd. Texas Red and Pacific Blue are registered trademarks of Thermo Fisher Scientific. Cy Dye is a registered trademark of Cytiva.

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CASPASE ASSAYS & APOPTOSIS INDUCERS

NucView® Caspase Substrates for Live Cells

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. Unlike FLICA assays that use irreversible inhibitors to label active caspases, NucView® caspase-3 substrates do not interfere with caspase activity, allowing monitoring of caspase activity in real time. NucView® can be fixed with formaldehyde after staining, but it cannot be used to stain fixed cells or tissues. For staining of fixed cells or tissues, see our TUNEL Assays.

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
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.

NucView® 488 Caspase-3 Enzyme Substrate in action. HT-1080 cells were treated with camptothecin and NucView® 488 Caspase-3 Substrate and imaged every 30 minutes for 24 hours on the IncuCyte® Zoom. Apoptotic cell nuclei fluoresce green as NucView® 488 dye is released by caspase-3 cleavage. Video courtesy of Essen Biosciences.

Apoptotic Jurkat cell stained with NucView® 488 (nucleus, green) and CF®647 Annexin V (cell surface phosphatidylserine, magenta).
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.
Untreated or staurosporine-treated MCF-7 cells stained with MitoView™ Blue, NucView® 530 (red), and CF®488A Annexin V (green). Healthy cells show mitochondrial staining with MitoView™. In apoptotic cells, mitochondrial localization of MitoView™ Blue is lost, and cells become positive for NucView® and Annexin V.

NucView® Caspase-3 Substrates and Kits

Catalog No.

Features

NucView® 405 Caspase-3 Substrate, 1 mM in DMSO 10405 Blue fluorescence for flow cytometry in the Pacific Blue® channel or microscopy with the 405 nm laser
NucView® 405 Caspase-3 Substrate, 1 mM in PBS 10407 NucView® 405 substrate in PBS, for DMSO-sensitive cell types
NucView® 488 Caspase-3 Substrate, 1 mM in DMSO 10402 Green fluorescent substrate validated in more than 100 cell types and 200 publications
NucView® 488 Caspase-3 Substrate, 1 mM in PBS 10403 NucView® 488 substrate in PBS, for DMSO-sensitive cell types
NucView® 530 Caspase-3 Substrate, 1 mM in DMSO 10406 Orange fluorescence for microscopy in the Cy®3 channel or flow cytometry in the R-PE channel
NucView® 530 Caspase-3 Substrate, 1 mM in PBS 10408 NucView® 530 substrate in PBS, for DMSO-sensitive cell types
NucView® 488 and MitoView™ 633 Apotosis Detection Kit 30062 NucView® 488 and far-red fluorescent MitoView™ 633 for the Cy®5 channel
NucView® 488 and RedDot™ 2 Apoptosis & Necrosis Kit 30072 NucView® 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 V 30067 NucView® 488 and red fluorescent Annexin V apoptosis probe
Dual Apoptosis Assay with NucView® 488 and CF®640R Annexin V 30073 NucView® 488 and far-red fluorescent Annexin V apoptosis probe

Pacific Blue is a registered trademark of Thermo Fisher Scientific. Cy Dye is a registered trademark of Cytiva.

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Other Caspase Substrates and Assays

In addition to our patented NucView® technology for detecting caspase-3 activity in live cells, Biotium also offers rhodamine 110 (R110)-based assay kits for fluorescence- or absorbance-based detection of caspase-3 or caspase-8 activity in cell lysates. The HTS versions of the R110-based homogenous caspase-3 and caspase-8 assay kits are optimized for high throughput screening by fluorescence microplate reader. We also offers a coumarin (AMC)-based blue fluorogenic substrate (Ac-DEVD-AMC) for measuring caspase-3 activity in cell lysates by fluorescence microplate reader.

Apoptosis Inducers/Inhibitors

Staurosporine is a broad range protein kinase inhibitor that induces apoptosis in cultured cells. It is useful as a positive control for many apoptosis assays. We also offer ionomycin, a calcium ionophore that has been shown to induce apoptosis through calpain activation. Caspase inhibitor Ac-DEVD-CHO is a competitive inhibitor of caspase-3 for use in cell lysates.


Other Caspase Assays and Apoptosis Inducers

Product

Catalog No.

Size

Caspase-3 DEVD-R110 Fluorometric & Colorimetric Assay Kit 30008-1 25 assays
30008-2 100 assays
Caspase-3 DEVD-R110 Fluorometric HTS Assay 30009-1 10 assays
30009-2 100 assays
30009-3 1000 assays
Ac-DEVD-AMC 10202 5 mg
Ac-DEVD-R110 10226 1 mg
Ac-DEVD-CHO Caspase-3 Inhibitor 10404-1 1 mg
10404 5 mg
Staurosporine 00025 100 ug
Ionomycin, Calcium Salt 59007 1 mg

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APOPTOSIS/NECROSIS KITS & ANNEXIN V

Dual Apoptosis & Necrosis Assay Kits

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. We offer kits with our bright and photostable CF®488A Annexin V and a variety of dead cell nucleic acid stains, including propidium iodide (PI), 7-AAD, Ethidium Homodimer III (EthDIII), and RedDot™2. These are membrane-impermeant fluorogenic nucleic acid dyes that stain necrotic and late apoptotic cells with damaged plasma membranes. EthDIII is a novel red fluorescent dead cell stain with higher affinity and quantum yield compared to PI, while RedDot™2 is a far-red dead cell stain for the Cy®5 channel. The Apoptotic, Necrotic, and Healthy Cells Quantitation Kit Plus also includes blue fluorescent Hoechst DNA dye for visualizing all cells by fluorescence microscopy. Also see our apoptosis and necrosis staining kit with NucView®488 Caspase-3 Substrate and the far-red dead cell stain RedDot™2 .

Annexin V conjugates are available with a wide selection of CF® dyes and other labels, including Near-IR CF® Dye Annexin V conjugates. We offer dead cell nucleic acid stains separately as well, including far-red fluorescent RedDot™2 far-red dye and green fluorescent NucSpot® 470 stain. Note: Apoptosis & Necrosis Staining kits and Annexin conjugates cannot be used with fixed cells or tissues. For fixed cell stains, see our TUNEL Assays.


Untreated and staurosporine-treated Jurkat cells stained with the Apoptotic, Necrotic and Healthy Cells Plus Kit. Apoptotic cells are stained green, necrotic/late apoptotic cells are stained red, and all cell nuclei are stained blue.

Apoptosis and Necrosis Staining Kits

Catalog No.

Features

CF®488A Annexin V and PI Apoptosis Kit 30061 • CF®488A stains apoptotic cells green for the FITC channel
• PI stains necrotic cells red for the Cy®3 or PE channels
• For microscopy or flow cytometry
CF®488A Annexin V and 7-AAD Apoptosis Kit 30060 • CF®488A stains apoptotic cells green for the FITC channel
• 7-AAD stains necrotic cells red for the PE-Cy®5 or FL3 channels
• For flow cytometry
Apoptosis & Necrosis Quantitation Kit Plus 30065 • CF®488A stains apoptotic cells green for the FITC channel
• EthD-III stains dead cells red for the Cy®3 or PE channels
• EthD-III has higher affinity and quantum yield than PI or 7-AAD
• For microscopy or flow cytometry
Apoptotic, Necrotic & Healthy Cells Quantitation Kit Plus 30066 • CF®488A stains apoptotic cells green for the FITC channel
• EthD-III stains dead cells red for the Cy® channel
• Hoechst stains all cell nuclei blue for the DAPI channel
• For microscopy
NucView®488 and RedDot™2 Apoptosis and Necrosis Kit 30072 • NucView®488 stains apoptotic nuclei green for the FITC channel
• RedDot™2 stains necrotic cells far-red for the Cy®5 or PE-Cy®5, APC, or FL3 channels
• For microscopy or flow cytometry
Annexin V Conjugates Multiple • Annexin V with our bright and photostable CF® dyes and a selection of other labels
Annexin V Conjugates, Azide-Free Multiple • Annexin V with our bright and photostable CF® dyes and a selection of other labels
• Lyophilized and preservative-free for real-time cell imaging
Annexin V Near-IR CF® Dye Conjugates Multiple • Annexin V conjugated to our superior Near-IR CF® dyes
• Lyophilized and preservative-free for in vivo use
Annexin V (His Tag) 29088 • Recombinant Annexin V (His Tag) expressed from E.coli
Annexin Binding Buffer 99902 • Concentrated Annexin Binding Buffer for cell staining
NucSpot® 470 Nuclear Stain 40083 • Green fluorescent dead cell stain
• Also useful as a nuclear-specific counterstain for fixed cells
RedDot™2 Far-Red Nuclear Stain 40061 • Far-red nuclear stain for dead cells or fixed cells
• Detect in the Cy®5 channel
Ethidium homodimer III 40051 • Red fluorescent dead cell stain
• Higher affinity and quantum yield than PI
Propidium Iodide 40017 • Widely used red fluorescent dead cell stain
7-AAD 40037 • Fluorescent dead cell stain for the PE-Cy®5/FL3 channel
NucSpot® Far-Red 40085 • Designed as an improved replacement for 7-AAD
• Less bleed into the PE-Texas Red® channel compared to 7-AAD

Cy Dye is a registered trademark of Cytiva.

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TUNEL ASSAYS & OTHER STAINS FOR FIXED CELLS OR TISSUES

CF® Dye Fluorescent TUNEL Staining Kits

TUNEL (terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling) is highly selective for the detection of apoptotic cells, but not necrotic cells or cells with DNA strand breaks resulting from irradiation or drug treatment. In this assay, TdT enzyme catalyzes the addition of labeled dUTP to the 3’ ends of cleaved DNA fragments. Fluorescent dye-conjugated dUTP can be used for direct detection of fragmented DNA in fixed cells or tissue sections. We offer TUNEL kits with dUTP conjugates of our bright and photostable CF® dyes with green, red, or far-red fluorescence. We also offer CF® dye dUTP Conjugates in a variety of colors.

Apoptotic cells in a section of involuting mouse mammary gland stained with CF®594 TUNEL Assay (red). Nuclei are counterstained blue with DAPI.

 

CF® Dye TUNEL Kits

Catalog No.

Ex/Em

Detection channel

CF®488A TUNEL Assay Apoptosis Detection Kit 30063 490/515 nm FITC
CF®594 TUNEL Assay Apoptosis Detection Kit 30064 593/614 nm Texas Red®
CF®640R TUNEL Assay Apoptosis Detection Kit 30074 642/662 nm Cy®5 or APC

Texas Red is a registered trademark of Thermo Fisher Scientific. Cy Dye is a registered trademark of Cytiva.

PathoGreen™ and Other Neuronal Histofluorescent Stains

PathoGreen™ Histofluorescent Stain is an anionic green fluorescent dye functionally similar to Fluoro-Jade® dyes. These dyes stain degenerating neurons and their processes in tissue sections and cultured cells. The mechanism of staining has not been determined, but it is proposed that the negatively charged dyes bind to positively charged polyamines or other molecules generated in dying neurons in response to a variety of different neurotoxic insults.

We also offer stains for the detection of amyloid in live or fixed neuronal tissues, including Congo Red, DCDAPH, and Thioflavin T.

Degenerating neurons in a section of mouse hippocampus stained with PathoGreen™.

PathoGreen™ & Other Histofluorescent Stains

Product

Catalog No.

Size

Features

PathoGreen™, 1000X in water 80027-5mL 5 mL Stains degenerating neurons in tissue sections or cultures with green fluorescence
80027-50mL 50 mL
Congo Red, High Purity Grade 80028 100 mg Colorimetric or fluorescent staining of amyloid plaques (Ex/Em 497/614 nm)
DCDAPH 80030 5 mg Detection of β amyloid in tissue sections and by near IR small animal imaging (Ex/Em 597/665 nm)
Thioflavin T, High Purity Grade 80033 100 mg Green fluorescent cell permeable fluorescent amyloid probe

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Faq

NucView® Caspase-3 Enzyme Substrates

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples. One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.  


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Bioscience kits The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples. Antibodies and other conjugates The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples. For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates). Chemicals, dyes, and gel stains Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage. If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples. Expiration date based on date of manufacture (DOM) If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance. Chemical products with special stability considerations: Esters Ester compounds include the following: • Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes • Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes • Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound. A note on CF® Dye succinimidyl ester stability Succinimidyl esters (SE) are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Many commercially available fluorescent dyes used for life science research are heavily sulfonated dyes which makes them particularly hygroscopic, worsening the hydrolysis problem. In addition, for several commercially available SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while the SE group in all of Biotium’s CF® Dyes is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF® Dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes. Maleimides, MTS and thiosulfate dyes Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions. Other reactive dyes Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information. Coelenterazines and D-luciferin Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution. Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution. Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced. D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.


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Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature or with an ice pack. These products may thaw without affecting product performance. When you receive the product, place it under the recommended storage conditions. Some products are shipped with blue ice packs as an extra precaution against high temperatures. The blue ice packs may be thawed upon arrival without affecting product performance. Products with recommended storage at ultra low temperature (-70°C) that also ship on dry ice should arrive frozen. If a product you received was shipped on dry ice and thawed during transit, please contact customer service at order@biotium.com. You can also download our Product Storage Statement here.


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