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NucRed™ Dead 647 ReadyProbes™ Reagent (TO-PRO-3 iodide) Invitrogen™

NucRed Dead 647 ReadyProbes Reagent is a cell impermeant stain that emits bright far-red fluorescence when bound to DNA. Since this reagent stains the cells without plasma membrane integrity it is extremely useful to measure viability and to measure cytotoxicity in kinetic live cell assays. NucRed Dead 647 reagent is also suitable for staining nuclei in fixed cell preparations and tissue sections.

Also available: TO-PRO-3 Iodide at 12.5X concentration (13.3 µM) in water with PowerLoad solubilizing agent and 1% DMSO.

• Exceptionally bright far-red fluorescence upon binding to DNA
• Rapid staining of damaged, dead, or fixed cells without wash steps
• Ready-to-use liquid formulation in convenient dropper bottle
• Stability at room temperature—keep handy at your scope or cell culture area

See other ReadyProbes reagents for cell staining
See other nuclear stains for imaging

Cell imaging applications
The far-red fluorescence emission makes it ideal for use in combination with blue, green, and red fluorophores, such as Hoechst, GFP/FITC, and Texas Red.

Suggestions for use
• NucRed Dead 647 ReadyProbes Reagent may be added directly to cells in full media, or buffer solutions.
• In most cases, 2 drops/mL and an incubation of 15 to 30 minutes will give bright nuclear staining; however, more or fewer drops can be added for optimal staining intensity.
• In most cases, staining intensity increases with time if cells are not washed prior to imaging.
• It is detected through a far-red filter, such as a Cy5 filter, or a Cy5 Light Cube for EVOS imaging systems.

FluoVolt™ Membrane Potential Kit Invitrogen™

The FluoVolt™ Membrane Potential Kit represents the next generation in voltage sensitive fluorescent probe-based kits. The FluoVolt™ probe brings together the best characteristics of the fast and slow response membrane potential fluorescent probes. The FluoVolt™ probe responds to changes in membrane potential in sub-milliseconds and displays a high magnitude of response.

Learn more about ion indicators including calcium, potassium, pH, and membrane potential indicators ›

Features of the FluoVolt™ Membrane Potential Probe include:

• Fast—responds to changes in membrane potential within sub-milliseconds
• High sensitivity—response range is typically 25% per 100 mV
• Emission/excitation works with standard FITC settings
• Can be used in imaging or patch clamp applications

Changes in membrane potential play a central role in many physiological processes, including nerve-impulse propagation, muscle contraction, and cell signaling. Potentiometric fluorescent probes are important tools for studying these processes and are generally characterized as slow- or fast-response probes.

The Best Characteristics of Slow- and Fast-Response Probes
Slow-response probes function by entering depolarized cells, binding to proteins or membranes, and exhibiting enhanced fluorescence. This membrane translocation event decreases the ability of these reporters to respond to changes in membrane potential and introduces a capacitive load that can affect cell health. However, these probes display a high magnitude of response; typically in the 1% per mV range.

Fast-response probes are molecules that change their structure in response to the surrounding electric field and detect transient (millisecond) potential changes. However, when compared to the slow-response probes, the fast-response probes have a magnitude of potential-dependent fluorescence change that is often small (2–10% fluorescence change per 100 mV).

The FluoVolt™ Membrane Potential Probe displays the best properties of the slow- and fast-response probes. The FluoVolt™ probe is a fast-response probe with a superior potential-dependent fluorescence response. The response is fast enough to detect transient (sub-millisecond) potential changes in excitable cells and generates a signal change in excess of 25% per 100 mV.

PowerLoad™ and Background Suppressor Solutions Also Included
For easy cell loading, the FluoVolt™ Membrane Potential Kit contains PowerLoad™ Concentrate. Due to the unique nature of the PowerLoad™ solution, it can be used in the presence of complete culture media, thus reducing the negative effects of replacing media or loading in serum-free media.

Baseline autofluorescence caused by components within growth media can be greatly reduced by the addition of the included Neuro Background Supressor. This solution has been specifically formulated for use with neuronal cells and will not cause osmotic shock. Additionally, the Neuro Background Suppressor has been used successfully with many different cell types to efficiently suppress background fluorescence without sacrificing the specific cellular fluorescence generated in the assay.

Vybrant™ DyeCycle™ Ruby Stain Invitrogen™

Vybrant® DyeCycle™ Ruby Stain is a cell permeable DNA dye that can be used for cell cycle analysis on a 488 nm or 635 nm red diode laser.

• Precise—accurate cell cycle analysis in living cells

• Safe—low cytotoxicity for cell sorting and additional live cell experiments

• Minimal compensation—easier multiplexing

• Simple, robust staining protocol

View a selection guide for all products related to cell cycle analysis of fixed and live cells in flow cytometry.

Precise

Successful cell cycle analysis requires a dye that is DNA selective and can stain cells in a homogeneous pattern minimizing fluorescence variability. The Vybrant® DyeCycle™ Ruby Stain is an ideal tool for DNA content analysis in living cells since the stain is cell permeable and, after binding double-stranded DNA, emits a fluorescent signal that is proportional to the DNA mass (see figure).

Low Cytotoxicity

Unlike stains that require high concentrations or have chemical structures that are toxic to cells, Vybrant® DyeCycle™ Ruby Stain exhibits relatively low cytotoxicity, allowing the possibility of sorting based on the phase of the cell cycle.

Minimal Compensation

Well-suited for the popular red 633 nm laser line, the Vybrant® DyeCycle™ Ruby Stain/DNA complex has fluorescence excitation and emission maxima of 638/686 nm, respectively (see figure). The spectral characteristics allow use of the red laser for cell cycle analysis and frees up other channels for additional parameters.

The red excitation and narrow emission of Vybrant® DyeCycle™ Ruby Stain make it ideal for multiplexing due to the limited spectral overlap with other common dyes (Alexa Fluor® 488, FITC, and RPE) and fluorescent proteins (Green Fluorescent Protein (GFP)).

Simple, Robust Staining Protocol

For cell analysis, simply prepare flow cytometry tubes containing 0.5 mL of cells suspended in complete media at a concentration of 5 × 105 cells/mL. To each tube, add 1 µL of Vybrant® DyeCycle™ Ruby Stain and mix well. Final stain concentration is 5 µM. Incubate at 37°C for 15–30 minutes, protected from light. Analyze without washing cells on a flow cytometer using 488 nm or 633/5 nm excitation and >670 nm emission.

RH 237 (N-(4-Sulfobutyl)-4-(6-(4-(Dibutylamino)phenyl)hexatrienyl)Pyridinium, Inner Salt) Invitrogen™

The fast-response potential-sensitive probe RH 237 can be used for imaging functional neurons.

TO-PRO™3 Ready Flow™ Reagent Invitrogen™

TO-PRO™3 Ready Flow™ Reagent is a bright and easy-to-use nucleic acid stain for distinguishing dead cells from live cells in flow cytometry assays. This dye has excitation/emission maxima of 642/661 nm, similar to Alexa Fluor™ 647 or allophycocyanin (APC).

• Stable at room temperature
• Convenient, ready-to-use format—no need to dilute, weigh, or pipette
• Rapid staining of dead cells without wash steps

TO-PRO 3 Ready Flow Reagent is a ready-to-use, direct-to-sample dead-cell stain that emits red fluorescence when bound to DNA. Add 2 drops form the dropper bottle directly into 1 x 106 cells, incubate, and analyze.

TO-PRO 3 Ready Flow Reagent is membrane impermeant and therefore excluded from viable cells. It binds selectively to double-stranded DNA. This dye has excitation/emission maxima of 642/661 nm.

DiR'; DiIC18(7) (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide) Invitrogen™

The near IR fluorescent, lipophilic carbocyanine DiOC18(7) ('DiR') is weakly fluorescent in water but highly fluorescent and quite photostable when incorporated into membranes. The sulfonate groups incorporated into this DiI analog improves water solubility. It has an extremely high extinction coefficient and short excited-state lifetimes (~1 nanosecond) in lipid environments. Once applied to cells, the dye diffuses laterally within the plasma membrane.

eBioscience™ Indo-1 AM Calcium Sensor Dye (UltraPure Grade) Invitrogen™

Indo-1 AM Calcium Sensor Dye is a membrane-permeable dye used for determining changes in calcium concentrations in the cell using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Once Indo-1 enters the cell, esterases cleave the AM group yielding a membrane-impermeable dye. Unbound Indo-1 has a peak emission at 475 nm. Upon binding calcium, the peak emission shifts down to 410 nm.

In flow cytometry this can be measured over time and can be represented as a ratio of the two emission wavelengths. To optimize the ratio between the two emissions, unbound Indo-1 fluorescence should be collected using a filter above 475nm (525 nm is a good option) while bound Indo-1 fluorescence should be collected using a filter below 400 nm. Because the emission profile of Indo-1 is broad, multicolor flow analysis using eFluor ™ 450, eVolve™ 605 or eVolve™ 655 is not possible, however fluorochrome conjugated antibodies utilizing the 488nm or 633 nm are quite compatible with Indo-1.

Molecular Weight: 1009.91
Peak Excitation: 346 nm
Peak Emission: 475 nm

Indo-1 AM should be reconstituted in high-quality, freshly opened DMSO. Recommended concentration: 1 mM.
Once reconstituted, it should be protected from light and stored at -20°C; avoid freeze-thaws.

Purity
>95% as determined by HPLC

Reported Application
Flow Cytometric Analysis, Microscopy, Immunohistochemical Staining

SYTO™ 85 Orange Fluorescent Nucleic Acid Stain - 5 mM Solution in DMSO Invitrogen™

The cell-permeant SYTO 85 orange fluorescent nucleic acid stain exhibits bright, orange fluorescence upon binding to nucleic acids. Because the staining pattern of the SYTO dyes in live cells may vary between cell types, we offer the SYTO Orange Fluorescent Nucleic Acid Stain Sampler Kit (S-11360) to enable researchers to find the most appropriate orange-fluorescent SYTO stain for their system.

ER-Tracker™ Red (BODIPY™ TR Glibenclamide), for live-cell imaging Invitrogen™

ER-Tracker™ Red dye is cell-permeant, live-cell stain that is highly selective for the endoplasmic reticulum (ER) that when stained using the protocol provided, the staining pattern is partially retained after fixation with formaldehyde. This stain consists of the green-fluorescent BODIPY® TR dye and glibenclamide. Glibenclamide (glyburide) binds to the sulphonylurea receptors of ATP-sensitive K+ channels which are prominent on ER; the pharmacological activity of glibenclamide could potentially affect ER function. Variable expression of sulphonylurea receptors in some specialized cell types may result in non-ER labeling.

LysoTracker™ Deep Red Invitrogen™

LysoTracker® Deep Red is a deep red-fluorescent dye for labeling and tracking acidic organelles in live cells.

Features of all LysoTracker® probes include:

• High selectivity for acidic organelles
• Effective labeling of live cells at nanomolar concentrations
• Live-cell imaging
• With excitation/emission of 647/668 nm, the same filter set can be used as used for Cy5 or MitoTracker® Deep Red

Simple, Highly Specific, One-Step Staining and Tracking of Acidic Organelles
The LysoTracker® probes consist of a fluorophore linked to a weak base that is only partially protonated at neutral pH. This allows LysoTracker® probes to freely permeate cell membranes enabling them to label live cells. LysoTracker® probes are highly selective for acidic organelles, and unlike dinitrophenyl (DNP), LysoTracker® probes do not require use of a second antibody for detection.

LysoTracker® probes are available in a variety of fluorescent colors.

Image-iT™ TMRM Reagent (mitochondrial membrane potential indicator) Invitrogen™

Healthy mitochondrial membranes maintain a difference in electrical potential between the interior and exterior of the organelle, referred to as a membrane potential. Tetramethylrhodamine, methyl ester (TMRM) is a cell-permeant dye that accumulates in active mitochondria with intact membrane potentials. If the cells are healthy and have functioning mitochondria, the signal will be bright. Upon loss of the mitochondrial membrane potential, TMRM accumulation will cease and the signal will dim or disappear. TMRM signal can be detected with fluorescence microscopy, flow cytometry, cell sorting, high-throughput screening, and high content analysis.

• For live-cell dynamic study of mitochondrial membrane potential (not fixable)
• Detectable with RFP/TRITC filter set (absorbance peak: 548 nm, emission peak: 574 nm)
• Mutiplexable with green, deep red, violet, and blue emission dyes, such as GFP, DAPI, Hoechst, HCS LipidTOX, and LysoTracker Deep Red

eBioscience™ Calcein AM Viability Dye (UltraPure Grade) Invitrogen™

Calcein AM is a membrane-permeable live-cell labeling dye. Upon entering the cell, intracellular esterases cleave the acetoxymethyl (AM) ester group, yielding the membrane-impermeable Calcein fluorescent dye. Apoptotic and dead cells with compromised cell membranes do not retain Calcein. Calcein is optimally excited at 495 nm and has a peak emission of 515 nm. For fluorescent microscopy, Calcein can be detected using the appropriate filter sets. For flow cytometric analysis, it can be excited off the blue laser line (488 nm) and detected using filters for FITC/Alexa Fluor™ 488 (530/30). For multicolor flow cytometry, concentrations in the range of 5 – 20 nM are recommended to ensure that compensation can be performed successfully. Co-staining with Annexin V or 7-AAD is recommended to allow the greatest resolution between live and dead/apoptotic cells. If single-color analysis is desired, improved resolution between live and dead/apoptotic cells may be obtained by using either Calcein Blue AM (cat. 65-0855) or Calcein Violet 450 AM (cat. 65-0854).

Molecular weight: 994.86
Peak excitation: 495 nm
Peak emission: 515 nm

Calcein AM should be reconstituted in high-quality, freshly opened DMSO.
Once reconstituted, it should be stored at -20°C with dessicant and used within a short period of time. Avoid freeze-thawing.

Purity
>95% as determined by HPLC.

Reported Application
Flow Cytometric Analysis, Microscopy

Vybrant™ DyeCycle™ Violet Ready Flow™ Reagent Invitrogen™

Vybrant DyeCycle Violet Ready Flow Reagent is a bright, easy-to-use, double-stranded DNA stain for cell cycle analysis in living cells using flow cytometry. It is excitable by a 405-nm laser with an emission peak at 437 nm.

• Stable at room temperature
• Convenient, ready-to-use format—no need to dilute, weigh, or pipette
• Accurate cell-cycle analysis in living cells
• Low toxicity for cell sorting and additional live cell experiments

Vybrant DyeCycle Violet Ready Flow Reagent is a ready-to-use, direct-to-sample DNA stain. Add 2 drops from the dropper bottle directly into 1 x 106 cells, incubate, and analyze.

As a cell-permeable dye, Vybrant DyeCycle Violet Ready Flow enters live cells and selectively binds dsDNA and emits a signal proportional to the DNA mass. The dye can be detected when excited with a 405-nm laser, and the emission captured at a peak of 437 nm. Vybrant DyeCycle Violet reagent is an ideal choice for DNA content analysis and can be included in multi-color cell cycle studies.

DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) Invitrogen™

A popular nuclear and chromosome counterstain, DAPI emits blue fluorescence upon binding to AT regions of DNA. Although the dye is cell impermeant, higher concentrations will enter a live cell.

Also available as a room-temperature-stable, ready-to-use solution: NucBlue Fixed Cell Ready Probes Reagent.
See other Ready Probes ready-to-use imaging reagents and accessories ›

SYTOX™ Green Nucleic Acid Stain - 5 mM Solution in DMSO Invitrogen™

SYTOX® Green nucleic acid stain is an excellent green-fluorescent nuclear and chromosome counterstain that is impermeant to live cells, making it a useful indicator of dead cells within a population.

Also available as a room-temperature-stable, ready-to-use solution: NucGreen Dead 488 Ready Probes Reagent.
See other Ready Probes ready-to-use imaging reagents and accessories ›

 Impermeant to live cells
 Excitation/Emission: 504/523 nm
 Use with 488 Argon-ion laser
 Works with mammalian cells and both Gram-positive and Gram-negative bacteria

Simple Viability Determination
SYTOX® Green allows quick determination of cell viability when using flow cytometers, fluorescence microscopes, fluorometers, or fluorescence microplate readers. It will not cross intact membranes, but will easily penetrate compromised membranes characteristic of dead cells. Because it exhibits >500-fold fluorescence enhancement upon binding nucleic acids, briefly incubating dead cells with SYTOX® Green will result in bright green fluorescence with an emission peak of 523 nm when excited by a 488 nm argon-ion laser or any other 450–490 nm source. The exceptional brightness of the signal produced in dead cells makes SYTOX® Green particularly useful for determining viability of not just mammalian cells, but both Gram-positive and Gram-negative bacteria.

Part of the Invitrogen Family of Cell Viability Stains and Assays
SYTOX® stains for dead cells are available in a variety of colors, including red, blue, and orange. Additionally, Invitrogen has incorporated SYTOX® stains into a number of assays for apoptosis, cell viability, and metabolism.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Related Link
•  Find out more about all SYTOX® Products.

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