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DRAQ5
is a highly cell permeable DNA-interactive agent, with fluorescence
signature extending into the infra-red region of the spectrum. It is a
good choice for nuclear staining of live cells, as it does not require a
UV laser source for excitation. DRAQ5 will work with most benchtop
confocal systems and due to its far-red emission it is spectrally ideally
compatible with GFP and FITC based fluors. DRAQ5 is a pure synthetic
compound with high affinity for DNA, it is stable at room temperature,
under normal lighting conditions, and it is soluble in water at
biologically compatible pH.
The unique characteristics of DRAQ5 offer exciting new advantages for
investigations using both fixed specimens and viable cells.
Main
features of DRAQ5 are:
Rapid staining of cells in seconds by simple addition of DRAQ5
to buffer or culture medium with no need to wash, prior to imaging.
Identification
of nuclear location and orientation.
High penetration through cell layers for confocal imaging studies.
Spectral compatibility with GFP and UV excited fluors in live
or fixed cells.
Excitation is possible using a wide range of convenient laser
light wave-lenghts eg. 488, 514, 568, 633 or 647nm.
Emission spectrum extends from 670nm into the low infra-red,
providing minimal overlap with the emission from visible range
dyes including GFP.
Does not
photobleach.
Provided in
convenient, ready to use formulation.
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| Figure:
Spectral characteristics of DRAQ5 : Excitation (left),
Emission (right). NOTE: DRAQ5 does absorb at 488nm, even
though this is not obvious from the spectra shown. |
Figure: Triple-label
false color images of interphase (a), anaphase (b) and
metaphase (c) breast tumor cells using a mitochondrion-specific
dye (red), a GFP-labelled fusion protein (green) and DRAQ5 used
as a vital DNA dye (arbitrarily assigned the color blue).
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| Product
Specific Literature References |
A novel
cell permeant and far red-fluorescing DNA probe, DRAQ5, for blood
cell discrimination by flow cytometry: P.J. Smith, et al.; J.
Immunol. Meth. 229, 131 (1999) Abstract
A novel deep red/low infrared fluorescent flow cytometric
probe, DRAQ5NO, for the discrimination of intact nucleated cells
in apoptotic cell populations: M. Wiltshire, et al.; Cytometry
39, 217 (2000) Abstract
Characteristics of a novel deep red/infrared fluorescent
cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by
flow cytometry, confocal and multiphoton microscopy: P.J.
Smith, et al.; Cytometry 40, 280 (2000) Abstract
Discrimination of DNA and RNA in cells by a vital fluorescent
probe: lifetime imaging of SYTO13 in healthy and apoptotic cells:
M.A. van Zandvoort, et al.; Cytometry 47, 226 (2002) Abstract
Histone H3 phosphorylation during Xenopus oocyte maturation:
regulation by the MAP kinase/p90Rsk pathway and uncoupling from
DNA condensation: A. Schmitt, et al.; FEBS Lett. 518,
23 (2002) Abstract
LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll
adapters TRAM and TRIF: K.A. Fitzgerald, et al.; J. Exp. Med. 198,
1043 (2003) Abstract
Optimization of three- and four-color multiparameter DNA
analysis in lymphoma specimens: M. Plander, et al.; Cytometry A
54, 66 (2003) Abstract
DRAQ5-based DNA content analysis of hematolymphoid cell
subpopulations discriminated by surface antigens and light scatter
properties: C.M. Yuan, et al.; Cytometry B Clin. Cytom. 58,
47 (2004) Abstract
Microtubule stress modifies intra-nuclear location of Msh2 in
mouse embryonic fibroblasts: N. Marquez, et al.; Cell Cycle 3,
662 (2004) Abstract
A combined in vitro/bioinformatic investigation of redox
regulatory mechanisms governing cell cycle progression: J.E.
Conour, et al.; Physiol. Genomics 18, 196 (2004) Abstract
Advanced microscopy solutions for monitoring the kinetics and
dynamics of drug-DNA targeting in living cells: R.J. Errington,
et al.; Adv. Drug Deliv. Rev. 57, 153 (2005) Abstract
DNA labeling in living cells: R.M. Martin, et al.;
Cytometry A 67, 45 (2005) Abstract
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| Code |
BOS-889-002 |
| Product
Name |
APOPTRAK [DRAQ5NO] |
Original Manufacturer: Biostatus
Limited
| Product
Specifications:
|
| MW: |
444 |
| Quantity: |
500 l |
| Concentration: |
1mM |
| Appearance: |
Dark blue solution,
odourless. |
| Formulation: |
Aqueous solution. |
| Long
Term Storage: |
+4C |
| Use/Stability: |
Stable solution.
Incompatible with strong oxidizing agents and strong bases.
Storage conditions should preclude storage at pH < 6.0.
Concentration of working solution: 10-50M sufficient for at
least 100-500 tests with a test sample volume of 100l. |
| Handling: |
Protect from light. Do
not freeze. |
| Hazard: |
CYTOTOXIC. TOXIC. |
Product description:
| Fluorometric
assays for cell death often incorporate the ability of intact
viable cells to remain unstained by a fluorescent probe, while
cells with compromised membranes allow entry of that probe and its
subsequent intracellular accumulation or binding (e.g. propidium
iodide binding to nuclear DNA) providing a positive staining for
the loss of cellular integrity. A new compound with the property
to consistently stain viable cells at a low level but with greater
up-take upon membrane disruption associated with cell death offers
new avenues to identify cellular subsets and transition states:
APOPTRAK benefiting from the unique fluorescence
characteristics of its parental compound DRAQ5.
Main Features & Advantages of APOPTRAK
- APOPTRAK is a N-oxide modification of the cell
permeable DNA probe DRAQ5 (Prod. No. BOS-889-001).
- Compared to DRAQ5, APOPTRAK shows reduced cellular
penetration properties, but retains the
advantageous chromophore and fluorescence characteristics of DRAQ5:
Emission wavelengths extending from 665 nm to beyond 780 nm (Em l
max of 700.5nm for 647nm
excitation) permit the use of APOPTRAK staining alone or its
incorporation into existing fluorometric
protocols for live/dead cell discrimination using visible range
fluorochromes or GFP-based probes.
- Importantly in contrast to propidium iodide APOPTRAK will
positively label intact cells at a low level (providing a positive
discrimination/reference signal while compromised membranes/cellular
integrity will result in a significantly increased signal).
- Flow cytometric multi-parameter analyses (e.g. APOPTRAK
versus light scatter versus surface binding of another probe such
as FITC-tagged Annexin V) permit assay validation and further
separation of subpopulations for live/dead (necrotic or apoptotic)
cell discrimination by flow cytometry.
- APOPTRAK has a lower DNA-affinity compared to DRAQ5 and
an increased propensity for cytosolic localisation when
incorporated into viable cells, significantly reducing its
cytotoxicity. Performance of APOPTRAK has been validated in
lymphoid cells although the wider applications for this probe are
yet to be explored for imaging and cell division studies.
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Figure:
Flow cytometry analysis of dying lymphoma cells using either
APOPTRAK alone or in combination with annexin V-FITC. APOPTRAK
staining of normal (untreated control) (left dot plot) versus
apoptotic human lymphoma cells populations (middle dot plot)
versus APOPTRAK staining in combination with Annexin V-FITC (Prod.
No. ALX-209-256) co-staining of cells undergoing drug-induced
apoptosis (right dot plot). |
| Product
Specific Literature References: |
| A novel deep red/low
infrared fluorescent flow cytometric probe, DRAQ5NO, for the
discrimination of intact nucleated cells in apoptotic cell
populations: M. Wiltshire, et al.; Cytometry 39, 217 (2000)
Abstract
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General Information:
| Background/Technical Information |
Application
Protocol:
Simplified protocols
can be developed that involve the staining of live cells (e.g.
10-50 M APOPTRAK for 15 min at room temperature) prior to
5-fold dilution of sample immediately before analysis. The
product presentation and suggested protocol have been optimised
for human lymphoma cell analyses alone (see Fig) and staining
schedules may need to be user-optimised for other cell types.
APOPTRAK may be incorporated into other probe-staining
protocols such as that described for fluorescein-labelled annexin
V binding (Vermes et al.; J. Immunol. Methods 184,
39 (1995) Abstract). For example:
Cells (4-6 x 105 cells/ml) are washed with cold
phosphate buffered saline (no sodium azide) and resuspended in the
same buffer at a concentration of 1 x 106 cells/ml
(alternatively -when staining with Annexin-V-FITC- resuspend in
1X binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM
CaCl2) at the same concentration of 1 x 106 cells/ml.
100l of the cell suspension is transferred to a polystyrene
round bottomed flow tube to which (optional:
5l of Annexin V-FITC) and 5l (final 50M working solution) of
APOPTRAK are added.
Samples are gently vortexed, then incubated for 15 min at room
temperature in the dark.
400l of flow sample buffer (or for AnnexinV-FITC staining:
1X binding buffer) is added to each tube and
samples analysed immediately by flow cytometry or held on ice for
a maximum of 1 h prior to analysis.
Subsequently dilutions with medium or FACS-staining solution
may be required for optimization. |
| General
Literature References: |
Characteristics
of a novel deep red/infrared fluorescent cell-permeant DNA probe,
DRAQ5, in intact human cells analyzed by flow cytometry, confocal
and multiphoton microscopy:
P.J. Smith, et al.; Cytometry 40, 280 (2000) Abstract;
A novel cell permeant and far red-fluorescing DNA probe, DRAQ5,
for blood cell discrimination by flow cytometry: P.J. Smith,
et al.; J. Immunol. Methods 229, 131 (1999) Abstract; |
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BOS-889-004
LIVE CELL STAINING WITH CyTRAK Orange FOR NUCLEUS AND CYTOPLASM DISCRIMINATION
Appropriate for:
Wide-field camera-based imaging
Confocal Laser Scanning Microscopy
CyTRAK
Orange (Prod. No. BOS-889-004) is a novel orange fluorescent dye (related to
DRAQ5 (Prod. No. BOS-889-001)) that stains both nucleus and cytoplasm. It is
water soluble and membrane permeant and can be used in LIVE cells in combination
with other common fluorophores, especially GFP fusions, FITC-labelled antibodies
and far-red dyes.
CyTRAK
Orange (Prod. No. BOS-889-004) can be used as a LIVE cell nuclear stain or as
a cell location dye to define the perimeter of the cell in a variety of
cell-based assays. It is highly compatible with existing protocols and can be
used across a wide range of instrumentation platforms.
Key
Features:
Spectral
- low visible range (457, 488, & 549nm)
excitation
- no excitation by
UV/red lasers
- orange emission
- no emission overlap with co-excited
"green" fluorophores
- no appreciable auto-fluorescence
Binding/Labelling
- differential intensity of nuclear &
cytoplasmic labelling
- non-crosslinking
- one fluorophore doing the work of two
Chemical
- assay buffer compatible
- low photobleaching
- membrane-permeant
- no DMSO required
- storable in fridge (2-8C)
Advantages:
Spectral
- can be used on a wide range of imaging
platforms
- single scan with
GFP/FITC/Cy2, then dual scan
with red excited dyes for more parameters
- no need to wash out
Binding/Labelling
- nuclear and cytoplasm locator
- visualizes nuclear and cytoplasmic boundaries
- for LIVE and FIXED cells
- useful in most cell types
Chemical
- no lyse or wash protocols needed
- easy to use
- rapid staining
- convenient aqueous solution
- real-time temporospatial event imaging in
LIVE cells
Applications:
Real-time
Live Cell Imaging
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nuclear/cytoplasmic membrane translocations
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nucleus/cytoplasm translocations
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cytoplasm/cytoplasmic membrane translocations
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cell/cell boundary interactions
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visualize nucleus and cytoplasm as separate compartments to follow any
GFP-tagged
temporospatial
change in live cells in a single acquisition from a 488nm excitation
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scan a far-red parameter (e.g. Mitotracker Far Red) by serial excitation
at 633nm
Cell-based
HCS Assays
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no need to use UV excitation
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will enter EVERY cell in the well
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emission profile allows single pass scanning
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not actively pumped
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view the whole cytoplasmic field
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visible range excitation no UV "white out" of 10-15% of library
compounds
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GFP-tags and cells located in one plate scan for higher throughput
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include MDR cells
Fixed
Cell Assays/Imaging/FISH
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stains nucleus and cytoplasm differently
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spectrally compatible with green (e.g. FITC) and far-red (Cy5) fluorescent
probes
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image cytoplasmic and nuclear markers co-incidentally
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Figure:
CyTRAK
Orange (Prod. No. BOS-889-004) co-excites at 488 nm with eGFP (shown), FITC
and Cy2 fluorophores avoiding the need for laser alignment in confocal
instruments when this combination is used. A separate series of parameters can
be detected with red-exciting fluorophores such as APC (shown) or APC-Cy7 since
CyTRAK Orange (Prod. No. BOS-889-004) is not excited at 633 or 647 nm.
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Figure:
CyTRAK Orange (Prod. No. BOS-889-004) has an
emission profile that permits the simultaneous acquisition of commonly used
"green" fluorphore signals (e.g. eGFP (shown) , FITC and Cy2) AND
far-red FRET dyes (e.g. PE- and APC-Cy7) combined with discrimination of both
the nuclear and cytoplasmic compartments
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Step
1: Label cells with 5μM CyTRAKOrange (Prod. No. BOS-889-004) for
20-30 min.
Step
2: Use high intensity threshold value for classification
of nuclear compartment.
Step
3: Then use lower intensity threshold to detect cell edge.
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Figure:
The GFP (green) compatibility of CyTRAKOrange (Prod.
No. BOS-889-004; red nuclei) provides rapid one-step cell feature discrimination
in both live-and fixed-cell assays.
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Manufacturer:
Manufactured by Biostatus Ltd. CyTRAK Orange is a
registered trademark of Biostatus Ltd.
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