Cell Viability and

Cell Death
Apoptosis Reagents
NucView® caspase-3 substrates ... p. 2
Other caspase substrates ... p. 3
Apoptosis inducers ... p. 3
Annexin V conjugates ... p. 4
Tunel assays ... p. 5
Mitochondrial membrane potential dyes ... pp. 6-7

Viability Assays
Live-or-Dye™ fixable dead cell stains ... p. 8
Cell viability assay kits ... p. 9

Cell Proliferation
ViaFluor® SE cell proliferation kits ... p. 9

Microbial Viability
PMAxx™ and PMA for viability PCR ... p. 10
Bacterial viability assay kits ... p. 11
Yeast viability assay kits ... p. 11
 NucView® Caspase-3 Substrates
Fluorescent caspase-3 detection in living cells
NucView® caspase-3 substrates are novel fluorescent probes that allow detection
of caspase-3/7 activity in intact cells in real-time. In contrast to other fluorogenic
caspase substrates or fluorescent caspase inhibitor based (FLICA) assays,
NucView® substrates can be used to detect caspase-3/7 activity in cells without
inhibiting apoptosis progression.

NucView® is made by attaching a nucleic acid binding dye to the caspase-3/7

substrate peptide sequence DEVD. This uncleaved substrate dye is unable to bind
Figure 1. Schematic showing the principle of intracellular
to DNA and remains non-fluorescent. Once the substrate is cleaved by caspase-3/7
caspase-3/7 detection using NucView® caspase-3
in apoptotic cells, it releases the high-affinity fluorescent DNA dye, which stains the substrates.
cell nucleus with bright and stable fluorescence signal.

NucView® 488
CF®594 Annexin V

NucView® 405 NucView® 530 Proven technology

NucView® 405
CF®488A Annexin V
NucView® 530
CF®488A Annexin V NucView® caspase detection
technology has been
extensively tested. NucView®
NucView® 488 488 Substrate has been:

• Published in over 200

scientific papers

• Validated in more than 70

different cultured cell lines

• Validated in more than 30

different primary cell types

NucView® features

• Real-time monitoring of
caspase-3/7 activity
NucView® Substrates
• Rapid, no-wash assay
Cat. # Size Description
• Available in 3 colors 10402-T 10 uL
NucView® 488 Caspase-3 Substrate, 1 mM in DMSO
10402 100 uL
• For flow cytometry, microscopy
10403-T 10 uL
or microplate reader NucView® 488 Caspase-3 Substrate, 1 mM in PBS
10403 100 uL
• Formaldehyde fixable 10405-T 10 uL
NucView® 405 Caspase-3 Substrate, 1 mM in DMSO
10405 100 uL
10407-T 10 uL
NucView® 405 Caspase-3 Substrate, 1 mM in PBS
10407 100 uL
10406-T 10 uL
NucView® 530 Caspase-3 Substrate, 1 mM in DMSO
10406 100 uL
10408-T 10 uL
NucView® 530 Caspase-3 Substrate, 1 mM in PBS
10408 100 uL
2 • www.biotium.com
 NucView® kits and other caspase assays

NucView® combination staining kits

Biotium also offers kits containing the NucView® 488 substrate together with other types of apoptosis and
viability dyes for convenient multi-parameter experiments.
• Dual Apoptosis Kit: NucView® 488 + Annexin V labeled with red or far-red dyes for co-detection of two
apoptotic events, caspase cleavage and phosphatidylserine (PS) translocation. For more Annexin V
conjugates see p. 4.
• Dual Apoptosis Kit: NucView® 488 + MitoView™ 633 for co-detection of two apoptotic events, caspase
cleavage and loss of mitochondrial membrane potential. For more information on MitoView™ see p. 6.
• Apoptosis/Necrosis Kit: NucView® 488 + RedDot™2 for concurrent measurement of caspase cleavage
(apoptosis) and loss of membrane integrity (necrosis).

Additional caspase substrates

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 NucView® Combination Kits and Other Caspase Substrates
caspase-3 and caspase-8 assay kits are optimized for high throughput and Inhibitors
screening by fluorescence microplate reader.
Cat. # Size Description
Biotium also offers a coumarin (AMC)-based blue fluorogenic substrate (Ac- 30067 50 assays
Dual Apoptosis Assay with NucView® 488
DEVD-AMC) for measuring caspase-3 activity in cell lysates by fluorescence Caspase-3 Substrate and CF®594 Annexin V
microplate reader. Dual Apoptosis Assay with NucView® 488
30073 50 assays
Caspase-3 Substrate and CF®640R Annexin V
NucView® 488 and MitoView™ 633 Apoptosis
Caspase inhibitor 30062 100 assays
Assay Kit
Ac-DEVD-CHO is a competitive inhibitor of caspase-3 for use in cultured cells NucView® 488 and RedDot™2 Apoptosis &
30072 100 assays
or cell lysates. Necrosis Kit
30029-T 25 assays NucView® 488 Caspase-3 Assay Kit for Live
Apoptosis inducers 30029 100 assays Cells
Staurosporine is a broad range protein kinase inhibitor that induces apoptosis 30008-1 25 assays Caspase-3 DEVD-R110 Fluorometric &
in cultured cells. It is useful as a positive control for many apoptosis assays. 30008-2 100 assays Colorimetric Assay Kit
We also offer ionomycin, a calcium ionophore that has been shown to induce 30009-1 10 assays
apoptosis through calpain activation.
30009-2 100 assays Caspase-3 DEVD-R110 Fluorometric HTS Assay
30009-3 1000 assays
30011-1 25 assays Caspase-8 IETD-R110 Fluorometric &
30011-2 100 assays Colorimetric Assay Kit
30012-1 10 assays
Figure 2. Apoptotic HeLa cell stained with 30012-2 100 assays Caspase-8 IETD-R110 Fluorometric HTS Assay
CF®488A Annexin V (green) and NucView®
30012-3 1000 assays
405 (cyan). See p. 4 for more information on
Annexin V conjugates. 10404-1 1 mg
Ac-DEVD-CHO Caspase-3 Inhibitor
10404 5 mg
10202 5 mg Ac-DEVD-AMC
00025 100 ug Staurosporine
59007 1 mg Ionomycin, calcium salt

www.biotium.com • 3
 Annexin V Conjugates and Kits
Annexin V conjugates CF®488A Annexin V Dual Apoptosis & Necrosis
Annexin V is a 35-36 kDa protein that has a high affinity for phosphatidylserine Assay Kits
(PS). During apoptosis, PS is translocated from the inner to the outer leaflet Biotium offers several staining kits that allow concurrent identification of
of the plasma membrane, where it can be stained by fluorescent conjugates late apoptotic and membrane-compromised necrotic cells by fluorescence
of Annexin V, for detection of apoptotic cells by flow cytometry (Fig. 1) or microscopy or flow cytometry. These dual staining kits all include green
fluorescence microscopy (Fig. 2). Biotium offers Annexin V conjugates and kits fluorescent CF®488A Annexin V paired with a dead cell-specific nucleic acid
featuring our exceptionally bright and photostable CF® dyes. For example, dye: either red fluorescent Ethidium Homodimer III (EthD-III), red fluorescent
our CF®488A green fluorescent Annexin V conjugate (Fig. 2) is much brighter propidium iodide (PI), or far-red fluorescent 7-AAD. EthD-III is a novel
and more photostable than the traditional FITC-Annexin V, allowing the use membrane-impermeant nucleic acid dye developed at Biotium with higher
of 10-fold less conjugate in staining. Our near-infrared CF® dye conjugates of affinity for DNA and higher fluorescence quantum yield than propidium iodide.
Annexin V are supplied lyophilized and preservative-free, and are suitable for in
vivo imaging.
The Apoptotic, Necrotic, and Healthy Cells Quantitation Kit also includes blue
DMSO 1 hr DMSO 2 hr DMSO 4 hr fluorescent Hoechst 33342 DNA dye for visualizing the healthy cells and dead
Staurosporine 1 hr Staurosporine 2 hr Staurosporine 4 hr
cells (Fig. 2).
FL4-H: CF640R Annexin V

FL4-H: CF640R Annexin V

FL4-H: CF640R Annexin V

Figure 2. Jurkat cells stained using the Apoptotic,

Necrotic & Healthy Cells Quantitation Kit Plus
after apoptosis induction with staurosporine.
Apoptotic cells stain with CF®488A Annexin V
(green), necrotic/late apoptotic cells stain with
FL1-H: NucView 488 FL1-H: NucView 488 FL1-H: NucView 488 EthDIII (red). All cells are stained with Hoechst
Figure 1. Jurkat cells were treated with staurosporine to induce apoptosis (pink), or with (blue).
DMSO as a negative control (blue) for the times indicated, then stained for 15 minutes at
room temperature with NucView® 488 Caspase-3 Substrate (FL1-H, x-axis) and CF®640R
Annexin V (FL4-H, y-axis) in cell culture medium prior to analysis using a BD LSRII flow
cytometer. See pp. 2-3 for more information on NucView® caspase-3 substrates.

Annexin V Conjugates Apoptosis and Necrosis Combination Kits

Annexin V Conjugate Ex/Em (nm) Cat. # Size Kit Name/Description Cat. # Size
CF®350 Annexin V, 50 ug/mL 347/448 29012 0.5 mL Apoptosis & Necrosis Quantitation Kit Plus with CF®488A
30065 50 assays
CF®405M Annexin V, 50 ug/mL 408/452 29009 0.5 mL Annexin V and EthD-III
CF®488 Annexin V, 50 ug/mL 490/515 29005 0.5 mL CF®488A Annexin V and 7-AAD Apoptosis Kit 30060 100 assays
CF®555 Annexin V, 50 ug/mL 555/565 29004 0.5 mL CF®488A Annexin V and PI Apoptosis Kit 30061 100 assays
CF®568 Annexin V, 50 ug/mL 562/583 29010 0.5 mL Apoptotic, Necrotic & Healthy Cells Quantitation Kit Plus
30066 50 assays
CF®594 Annexin V, 50 ug/mL 593/614 29011 0.5 mL with CF®488A Annexin V, EthD-III and Hoechst
CF®633 Annexin V, 50 ug/mL 630/650 29008 0.5 mL
CF®640R Annexin V, 50 ug/mL 642/662 29014 0.5 mL
CF®647 Annexin V, 50 ug/mL 650/665 29003 0.5 mL
CF®660R Annexin V, 50 ug/mL 663/682 29069 0.5 mL Dual apoptosis assay kits
CF®680R Annexin V, lyophilized 680/701 29070 25 ug Annexin V conjugated to our deep red CF®594 or far-red CF®640R dyes is
CF®680 Annexin V, lyophilized 681/698 29007 25 ug offered together with NucView® 488 Caspase-3 Substrate for simultaneous
CF®750 Annexin V, lyophilized 755/777 29006 25 ug detection of caspase-3 activity and phosphatidylserine exposure by
CF®770 Annexin V, lyophilized 770/797 29046 25 ug fluorescence microscopy or flow cytometry (see p. 2 for more information on
CF®790 Annexin V, lyophilized 784/806 29047 25 ug NucView® substrates).
CF®800 Annexin V, lyophilized 797/816 29078 25 ug
FITC Annexin V, 50 ug/mL 490/525 29001 0.5 mL
Texas Red Annexin V, 50 ug/mL 583/603 29002 0.5 mL Dual Apoptosis Kits
496, 546, 29045-100 uL 20 assays
R-PE Annexin V Kit Name Cat. # Size
565/578 29045-500 uL 100 assays
29057-100 uL 20 assays Dual Apoptosis Assay with NucView® 488 and CF®594
APC Annexin V 633, 640/660 30067 50 assays
29057-500 uL 100 assays Annexin V
Biotin Annexin V, 50 ug/mL N/A 29013 0.5 mL Dual Apoptosis Assay with NucView® 488 and CF®640R
30073 50 assays
5X Annexin V Binding Buffer N/A 99902 15 mL Annexin V

4 • www.biotium.com
 TUNEL Assays and dUTP conjugates
CF® dye TUNEL kits and dUTP conjugates
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 TUNEL Assays and dUTP Conjugates
for direct detection of fragmented DNA by fluorescence microscopy or flow Product Name Ex/Em (nm) Cat. # Size
cytometry.
CF®488A TUNEL Assay Kit 490/515 30063 50 reactions

Biotium offers dUTP conjugated to a range of CF® dye colors for fluorescent CF®594 TUNEL Assay Kit 593/614 30064 50 reactions
TUNEL labeling, as well as direct TUNEL kits with green fluorescent CF®488A, CF®640R TUNEL Assay Kit 642/662 30074 50 reactions
red fluorescent CF®594, and far-red fluorescent CF®640R. We also supply 40004-T 5 nmol
dUTP conjugated to classic fluorophores and biotin. Visit www.biotium.com CF®405S-dUTP 404/431
40004 25 nmol
to see our selection of CF® dye conjugated streptavidin, as well as other
40100-T 5 nmol
nucleotide conjugates for probe labeling. CF®405M-dUTP 408/452
40100 25 nmol
40100-T 5 nmol
CF®488A-dUTP 490/515
40008 25 nmol
A. Control B. Staurosporine 40008-T 5 nmol
CF®543-dUTP 541/560
40002 25 nmol
40002-T 5 nmol
CF®568-dUTP 562/583
40005 25 nmol
40005-T 5 nmol
CF®594-dUTP 593/614
40006 25 nmol
40006-T 5 nmol
CF®640R-dUTP 642/662
40007 25 nmol
Figure 1. Jurkat cells labeled using the CF®488A TUNEL Assay Apoptosis
Detection Kit after no treatment (A) or apoptosis induction with 1 uM 40007-T 5 nmol
CF®680R-dUTP 680/701
staurosporine for 3 hours (B). Nuclei are counterstained with DAPI (blue). 40003 25 nmol
Cyanine 555-dUTP 550/570 40064 25 nmol
Cyanine 647-dUTP 650/670 40065 25 nmol
DEAC-dUTP 426/480 40059 25 nmol
5-TAMRA-dUTP, 1 mM solution 553/577 40001 25 uL
Fluorescein-12-dUTP 494/521 40063 25 nmol
5-AA-dUTP, 10 mM N/A 40020 100 uL
5-AA-dUTP, lyophilzed N/A 40020-1 1 mg
Digoxigenin-dUTP N/A 40078 25 nmol
5-Bromo-dUTP, 10 mM solution N/A 40025 25 uL
Biotin-11-dUTP, 1 mM solution N/A 40029 50 uL
Biotin-11-dUTP, lyophilized N/A 40029-1 50 ug
Biotin-16-dUTP, 1 mM solution N/A 40022 50 uL

Figure 2. TUNEL staining of paraffin sections of rat Biotin-16-dUTP, lyophilized N/A 40022-1 50 ug
mammary gland five days post-weaning (ApopTag® Biotin-20-dUTP, 1 mM solution N/A 40030 50 uL
positive control slides, MilliporeSigma) using
CF®594-dUTP (red). Nuclei are stained with DAPI Biotin-20-dUTP, lyophilized N/A 40030-1 50 ug
(blue).

www.biotium.com • 5
 Mitochondrial Membrane Potential Dyes
MitoView™ Dyes
Loss of mitochondrial membrane potential is a hallmark for apoptosis. Biotium offers the MitoView™ 633 dye for membrane potential-sensitive staining of
mitochondria by microscopy or flow cytometry. We also offer MitoView™ Blue, 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.

A. MitoView™ 405 B. MitoView™ 488

Figure 5. Flow cytometry analysis of Jurkat cells treated with CCCP

C. MitoView™ 633 D. MitoView™ 720 to depolarize the mitochondrial membrane or staurosporine to induce
apoptosis, resulting in decreased MitoView™ 633 staining.

Figure 4. HeLa cells stained with A. MitoView™ Blue, B. MitoView™ Green, C. MitoView™
633, or D. MitoView™ 720.

MitoView™ Dyes
Potential-
Cat. # Product Ex/Em (nm) Unit Size
dependent?
70070-T 50 ug
MitoView™ 405 398/440 Partial†
70070 20 x 50 ug
70054-T 50 ug
MitoView™ Green 490/523 No
70054 20 x 50 ug
70055-T 50 ug
MitoView™ 633 622/648* Yes
70055 20 x 50 ug
70068-T 50 ug
MitoView™ 720 720/758 nm** Partial†
70068 20 x 50 ug
*MitoView™ 633 also has visible red fluorescence in the Cy®3/rhodamine channel. It is not recommended for
imaging with other visible red probes.
**While optimal for Cy®7 settings, MitoView™ 720 is bright enough to be imaged in the Cy®5 channel, and can
be combined with visible red fluorescent probes.
†
Dyes with partial mitochondrial membrane potential dependence localize to the cytoplasm after mitochondrial
depolarization, but still retain fluorescence.

6 • www.biotium.com
 Mitochondrial Membrane Potential and Cellular Glutathione
JC-1 and other mitochondrial 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 plate reader.

We also offer a selection of classic potentiometric mitochondrial stains, including TMRE, TMRM, and DiIC1(5), in a variety of fluorescent colors.

MCB Glutathione Detection Kit

Diminished cellular glutathione (GSH) level occurs during apoptosis due to GSH efflux from mitochondria. Monochlorobimane (MCB), which reacts with thiols to
form a blue fluorescent product, allowing fluorometric quantitation of GSH in cell lysates.

MCB MCB-glutathione conjugate

non-fluorescent Ex/Em: 380/461 nm

Figure 6. MCB glutathione assay principle.

Figure 7. Jurkat cells were treated with DMSO (Control) or induced to undergo
apoptosis by treatment with 1 uM staurosporine for 5 hours. Glutathione levels
were measured using the MCB Glutathione Detection Kit by fluorescence
microplate reader.

Other Mitochondrial Dyes

Mitochondrial Membrane
Dye Color Ex/Em Cat. # Size
Potential Dependent?
JC-1, chloride salt Green/ 510/527 nm (cytoplasm) Two-color detection mitochondria 70011 5 mg
Red 585/590 nm (polarized polarization/depolarization
mitochondria)
JC-1, iodide salt Green/ 510/527 nm (cytoplasm) Two-color detection mitochondria 70014 5 mg
Red 585/590 nm (polarized polarization/depolarization
mitochondria)
Rhodamine 123 Green 505/534 nm Yes 70010 50 mg
TMRE Red 548/573 nm Yes 70016 25 mg
TMRE, 2 mM in DMSO Red 548/573 nm Yes 70005 0.5 mL
TMRM Red 548/573 nm Yes 70017 25 mg
DASPEI Red 461/589 nm Yes 70018 100 mg
DiIC1(5) Far-red 638/658 nm Yes 70015 100 mg

Assay Kits
Kit Name and Components Color Ex/Em Assay Cat. # Size
NucView® 488 and MitoView™ 633 Green/ 500/530 nm (caspase-3) Two color detection of caspase-3 30062 100
Apoptosis Kit Red 622/648 nm (polarized activity and mitochondrial potential assays
mitochondria)
JC-1 Mitochondrial Membrane Green/ 510/527 nm (cytoplasm) Two-color detection mitochondria 30001 100
Potential Detection Kit Red 585/590 nm (polarized polarization/depolarization assays
mitochondria)
MCB Glutathione Detection Kit Blue 394/490 nm Detection of cellular glutathione 30019 100
assays
www.biotium.com • 7
 Live-or-Dye™ Fixable Viability Stains
Live-or-Dye™
Live-or-Dye™ Fixable Viability Staining Kits are designed for discrimination between live and dead cells by flow cytometry and microscopy. Dead cell stains are
useful probes to include when analyzing cell surface protein expression by flow cytometry (Fig. 2), because they allow intracellular fluorescence signal from dead
cells with permeable plasma membranes to be excluded from analysis. Live-or-Dye™ Fixable Viability Stains are cell membrane impermeable; they enter dead
cells that have compromised membrane integrity and covalently label free amines on intracellular proteins. Live-or-Dye™ Fixable Viability Staining Kits can also be
used to discriminate live from dead cells by microscopy (Fig. 1A). Live-or-Dye™ labeling is extremely stable, allowing the cells to be fixed and permeabilized without
loss of fluorescence or dye transfer between cells.

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 (Fig. 1B). The dye is able to enter into dead
cells that have compromised membrane integrity and covalently label the cell nucleus, allowing for clear differentiation of live and dead cells by either microscopy or
flow cytometry. Unlike other commonly used nuclear stains such as propidium iodide or DRAQ®7, NucFix labeling is extremely stable, allowing the cells to be fixed
and permeabilized without loss of fluorescence or dye transfer between cells.

Live-or-Dye™ Fixable Stains for Dead Cells Live-or-Dye™ NucFix Red

• Affordable: Lower cost than Thermo Fisher

• Unique, nuclear dead cell stain
LIVE/DEAD® stains
• Choice: 8 bright colors across the spectrum • Fixable, unlike other commonly used nuclear
• Specific: No staining of live cells stains such as Propidium Iodide or DRAQ®7

• Fixable: No loss of brightness after fixation

• Versatile: For flow cytometry and microscopy

A. Live-or-Dye™ 488/515 B. Live-or-Dye™ NucFix Red

Figure 2. Discrimination of live and dead Jurkat cells by flow cytometry using Live-or-Dye™
Figure 1. HeLa cells were treated with ethanol to kill a fraction of the Fixable Viability Stains. Heat killed cells (solid peaks) showed much higher fluorescence
cells. The cells were stained with A) Live-or-Dye™ 488/515 or B) Live- intensity compared to live cells (white peaks), allowing the two populations to be clearly
or-Dye™ NucFix Red. Nuclei were counterstained with Hoechst. distinguished.

Live-or-Dye™ Kits
Validated applications
Cat. # Cat. # Emission Abs/Em
Kit Name Laser Line (FC=flow cytometry;
200 Reactions 50 Reactions Filter Maxima
M=microscopy)
Live-or-Dye™ 350/448 32002 32002-T 355 nm DAPI or Violet 347/448 nm FC
Live-or-Dye™ 405/452 32003 32003-T 405 nm Pacific Blue 408/452 nm FC
Live-or-Dye™ 405/545 32009 32009-T 405 nm AmCyan 395/545 nm FC
Live-or-Dye™ 488/515 32004 32004-T 488 nm FITC 490/515 nm FC, M
Live-or-Dye™ 568/583 32005 32005-T 488 or 561 nm PE 562/583 nm FC, M
Live-or-Dye™ 594/614 32006 32006-T 488 or 561 nm PE-Texas Red® 593/614 nm FC, M
Live-or-Dye™ 640/662 32007 32007-T 633 or 640 nm APC 642/662 nm FC, M
Live-or-Dye™ 750/777 32008 32008-T 633 or 640 nm APC-Cy7 755/777 nm FC
Live-or-Dye™ NucFix Red 32010 32010-T 488 or 532 nm PE-Texas Red® 520/610 nm FC, M
LIVE/DEAD and Texas Red are registered trademarks of Thermo Fisher Scientific. DRAQ is a registered trademark of Biostatus, Ltd.
8 • www.biotium.com
 Cellular Viability and Proliferation Assays
Calcein AM Cell Viability Assay Resazurin, MTT, and XTT Viability Assays
Calcein AM is a non-fluorescent, membrane permeable compound. Esterase MTT, XTT, and resazurin (alamarBlue®) are reduced by mitochondrial metabolic
activity in the cytoplasm of viable cells converts calcein AM to the green activity to yield colored or fluorescent products, and thus are useful for
fluorescent, membrane-impermeant compound calcein, which is retained in assaying cell viability and quantitating cell number. MTT and XTT are reduced
viable cells with intact plasma membranes. The Viability/Cytotoxicity Assay Kit to colored formazin salts that can be measured by absorbance. MTT generates
for Animal Live & Dead Cells pairs calcein AM with the dead cell dye Ethidium an insoluble formazin salt, requiring cell lysis before the absorbance can be
Homodimer III for quantitation of live and dead cells. measured, while XTT does not require cell lysis for measurement. Resazurin
is a non-fluorescent blue dye that is reduced to the pink fluorescent compound
A. B.
resorufin, which can be measured by fluorescence or absorbance.
1000

ViaFluor® SE Cell Proliferation Kits

Fluorescence

100

ViaFluor® SE Cell Proliferation Kits diffuse passively into cells covalently

10 label intracellular proteins throughout the cell. They can be used as cell-filling
stains for imaging morphology, or to track cell division by dye dilution. With
1 each cell division, daughter cells inherit roughly half of the fluorescent label,
allowing the number of cell divisions to be detected by the appearance of
200 600 1800 5400 16200
Cell Number
successively dimmer fluorescent peaks on a flow cytometry histogram. Staining
Figure 1. A. Quantitation of HeLa cell numbers in a 96-well assay plate using the Calcein AM is formaldehyde fixable.
Cell Viability Assay Kit. B. Live and dead HeLa cells stained with the Viability/Cytotoxicity Assay
for Animal Live & Dead Cells. Live cells are stained green, dead cells are stained red. ViaFluor® CFSE is the classic cell proliferation dye, detected in the FITC
channel. Biotium created ViaFluor® 488, a new improved green dye that is less
toxic, less leaky and more fixable than CFSE. We also offer blue fluorescent
PathoGreen™ Histofluorescent Stain ViaFluor® 405 for the violet laser. ViaFluor® 405 has improved brightness and
PathoGreen™ Histofluorescent Stain is an anionic green fluorescent dye less toxicity than CFSE.
functionally similar to Fluoro-Jade® dyes. These dyes stain degenerating
neurons and their processes in fixed brain sections and cultured neurons.
The dyes stain apoptotic and necrotic neurons after exposure to a variety of
neurotoxic insults. The mechanism of neuronal staining by anionic fluorescent
dyes has not been determined. It has been proposed that the negatively
charged dyes bind to positively charged polyamines or other molecules
specifically generated in dying neurons.

ATP-Glo™ Bioluminometric Cell Viability Assay

This assay takes advantage of the ATP-dependent oxidation of D-Luciferin
by Firefly luciferase and the resulting production of light in order to assess Figure 3. Cell division tracking in Jurkat cells over successive days. Cells were labeled
with ViaFluor® 405 (left) or ViaFluor® 488 (right) on day 0, and analyzed by flow
the amount of ATP in a cell culture, which is proportional to the number of cytometry on each following day. Each successively dimmer peak represents one cell
viable cells. The ATP-Glo™ kit can be used to detect as little as a single cell division. Unstained cells are in gray.
or 0.01 picomole of ATP, with signal linearity for ATP detection within 6 orders
of magnitude. This is a flash-type assay designed for detection using a single Cellular Viability Assays
sample luminometer or a luminometer with an injector in 96-well plate format. Kit Name Cat. # Unit Size
The luminescent signal is stable for up to one minute.
Viability/Cytotoxicity Assay Kit for Animal 30002-T 150 assays
Live & Dead Cells 30002 300 assays
Calcein AM Cell Viability Assay Kit 30026 1000 assays
10000
PathoGreen™ Histofluorescent Stain, 80027-5mL 5 mL
1000 1000X in water 80027-50mL 50 mL
Luminescence

100 30025-1 25 mL (2500 assays)

Resazurin Cell Viability Assay Kit
10 30025 100 mL (10,000 assays)
1 MTT Cell Viability Assay Kit 30006 1000 assays
1 10 100 1000 10000
XTT Cell Viability Assay Kit 30007 1000 assays
Cell Number
30020-T 50 assays
Figure 2. A section of mouse Figure 3. Quantitation of 10-fold serial dilutions ATP-Glo™ Bioluminometric Cell Viability
30020-1 200 assays
hippocampus stained with PathoGreen™ of Jurkat cells in suspension using ATP-Glo™ Assay Kit
Histofluorescent Stain. Degenerating Bioluminetric Cell Viability Assay using a single- 30020-2 1000 assays
neurons are stained green. sample luminometer. ViaFluor® 405 SE Cell Proliferation Kit 30068 1 kit
ViaFluor® 488 SE Cell Proliferation Kit 30086 1 kit
ViaFluor® CFSE Cell Proliferation Kit 30050 1 kit
alamarBlue is a registered trademark of Morphosys UK Ltd.
Fluoro-Jade is a registered trademark of Histo-Chem, Inc. www.biotium.com • 9
 PMAxx™ and PMA Dyes for Viability PCR
Viability PCR (v-PCR)
Viability PCR is a powerful technology for the sensitive and rapid detection of viable microorganisms. Unlike time-consuming culturing procedures, qPCR is a fast
and sensitive method of detection. However, normal qPCR does not distinguish between live and dead cells. With v-PCR using PMAxx™ or PMA, you get the
speed, sensitivity and specificity of PCR, plus quantifiable viability. And because no culturing is required, you can even detect viable but not culturable (VBNC)
bacteria.

How does v-PCR work?

PMAxx™ and PMA are photoreactive dyes with high affinity for DNA. The dyes intercalate into dsDNA and
form a covalent linkage upon exposure to intense visible light. PMAxx™ and PMA inhibit PCR amplification
of modified DNA templates by a combination of removal of modified DNA during purification and inhibition
of template amplification by DNA polymerases. Because PMAxx™ and PMA are cell membrane-
impermeable, when a sample containing both live and dead bacteria is treated with dye, only dead bacteria
with compromised cell membranes are susceptible to DNA modification (Fig. 1). In a real-time PCR
reaction, dead cell DNA will show delayed amplification and higher Ct than live cells. In a mixed population, Figure 1. Mechanism of PMA and PMAxx™. The cell membrane-
v-PCR permits quantitation of cell viability. The v-PCR technology can be applied not only to bacteria but to impermeable dyes PMA and PMAxx™ selectively and covalently
modify DNA from dead bacteria with compromised membranes
other cell types as well. while leaving DNA from viable cells intact.

PMAxx™ vs PMA
Since Biotium developed PMA in 2006, it has been used extensively in many applications and in hundreds of publications. However there are cell types and
conditions in which dead cell DNA inactivation by PMA is incomplete, which could lead to false positive results. After extensive testing, the scientists at Biotium have
invented a new dye called PMAxx™ that has the same spectral properties and is even more effective than PMA at live/dead discrimination by viability PCR (Fig. 2).

v-PCR LED Photolysis Devices

PMA-Lite™:
• Holds up to 18
microcentrifuge tubes
• Bright, long-lasting blue
LED lights
• Fan ensures temperatures
lower than 37oC. Figure 2. Live or heat-killed Bacillus subtilis were treated with PMA or
PMAxx™, followed by exposure with the PMA-Lite™ and DNA purification.
Fast EvaGreen® qPCR Master Mix was used to amplify a 500-bp fragment
of B. subtilis DNA. qPCR of dead cells treated with PMAxx™ showed a
Glo-Plate™ Blue: significant further delay (>7 Ct) compared to dead cells treated with PMA.
• Flat illumination surface fits
microplates Ordering Information
• Bright, long-lasting blue Cat. # Product Name Unit Size
LED lights
• Surface stays cool during 40069 PMAxx™ dye, 20 mM in dH2O 100 uL
light exposure. 40013 PMA dye 1 mg
40019 PMA dye, 20 mM in dH2O 100 uL
E90002 PMA-Lite™ LED Photolysis Device 1 device
Strain-specific v-PCR kits available for: E90004 Glo-Plate™ Blue 1 device
• Salmonella enterica 31038 PMA Enhancer for Gram-Negative Bacteria 16 mL

• Escherichia coli 31033 Real-Time Bacterial Viability Kit-Salmonella (InvA) 200 assays
31034 Real-Time Bacterial Viability Kit-M. tuberculosis (groEL2) 200 assays
• Escherichia coli O157:H7
31035 Real-Time Bacterial Viability Kit-Staph. aureus (nuc) 200 assays
• Listeria monocytogenes
31036 Real-Time Bacterial Viability Kit-MRSA (mecA) 200 assays
• Legionella pneumophila
31050 Real-Time Bacterial Viability Kit-E. coli (uidA) 200 assays
• Mycobacterium tuberculosis
31037 Real-Time Bacterial Viability Kit-E. coli O157:H7 (Z3276) 200 assays
• Staphylococcus aureus
31051 Real-Time Bacterial Viability Kit-Listeria monocytogenes (hly) 200 assays
• Methicilin resistant Staphylococcus aureus (MRSA)

31053 Real-Time Bacterial Viability Kit-Legionella pneumophila (mip) 200 assays

10 • www.biotium.com
 Microbial Viability Assays
Bacteria Viability Dyes and Kits
Live-or-Dye™ Fixable Viability Staining Kits utilize dead-cell-specific fixable dyes (Fig. 1). They are good for flow cytometry and microscopy and available in 9
bright, photostable colors. See p. 8 for more information on Live-or-Dye™ stains. CTC is a fluorescent dye that has been used to evaluate the respiratory activity
in bacteria. Healthy cells will reduce CTC into an insoluble red product. The Viability/Cytotoxicity Assay Kit for Bacteria Live and Dead Cells features dual staining:
DMAO for live cells, and EthD-III for dead cells (Fig. 2).

Combination Gram Stain and Viability Kits

It can be useful to distinguish live bacteria from dead, as well as Gram+ from Gram-, in the same sample. Our combination bacterial viability and fluorescent gram
staining kits can help (Fig. 3). Our fluorescent gram stains utilize fluorescently-labeled wheat germ agglutinin (WGA) to selectively stain the cell walls of gram-
positive bacteria.

Figure 1. Live and heat-killed E. coli Figure 2. Live and heat-killed E. coli Figure 3. Bacterial Viability and Gram
stained with Live-or-Dye™ 568/583 stained with DMAO, marking live cells Stain Kit. CF®488A-WGA, EthD-III and
(red) and DAPI (blue). (green) and EthD-III, marking dead DAPI.
Bacteria Viability Stains cells (red).

Cat. # Product Description

10063 CTC (5-Cyano-2,3-ditolyl tetrazolium chloride) Forms insoluble red product in respiring cells

30027 Viability/Cytotoxicity Assay Kit for Bacteria Live and Dead Cells DMAO to stain all cells and EthD-III for dead cells

32001 Bacterial Viability and Gram Stain Kit WGA for gram stain, EthD-III for dead cells, and DAPI for all cells

Yeast Viability Dyes and Kits

It is often useful to distinguish live yeast cells from dead, or identify cells that are metabolically active. Our selection of yeast viability dyes and kits can help.
• Live-or-Dye™ Fixable Viability Staining Kits: Fixable and dead-cell-specific. Good for flow cytometry and microscopy. Available in 9 bright, photostable colors.
Note: the NucFix™ Red variation is not nucleus-specific in yeast. See p. 8 for more information on Live-or-Dye™ stains.
• ViaVac™ Red/Green: A vacuolar cell vitality dye. Passively diffuses into cells and gives a nonspecific green staining pattern. In metabolically active yeast, the
dye is actively transported into the vacuole where it stains intravacuolar tubules bright red.

Figure 4. Yeast Vitality Staining Kit, Figure 5. Yeast Viability Staining Kit, Figure 6. Yeast Fixable Live/Dead
ViaVac™ Red/Green (red, healthy CF®-ConA (red, cell wall) and Live-or- Staining Kit, Thiazole Orange (green,
vacuolar tubules) and Calcofluor White Dye™ (green, dead cell cytoplasm). all cells) and Live-or-Dye™ 568/583
(blue, cell wall). (red, dead cell cytoplasm). Overlapping
signal appears yellow.
Yeast Viability Stains
Cat. # Product Description

29068 ViaVac™ Red/Green, 10 mM in DMSO Yeast vital dye

31062 Yeast Vitality Staining Kit ViaVac™ Red/Green and Calcofluor White

31063 Yeast Viability Staining Kit CF®-ConA and Live-or-Dye™ combinations

31064 Yeast Fixable Live/Dead Staining Kit Thiazole Orange and Live-or-Dye™ 568/583 www.biotium.com • 11
 Biotium, Inc.

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