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DiIC16(3) (1,1'-Dihexadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate) (Invitrogen™)

The red-orange fluorescent, lipophilic carbocyanine DiIC16(3) is a shorter-chain DiI (DiIC18(3)) analog that may incorporate into membranes more easily than DiI. It is weakly fluorescent in water but highly fluorescent and quite photostable when incorporated into membranes. 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.

NeuroTrace™ CM-DiI Tissue-Labeling Paste (Invitrogen™)

NeuroTrace® CM-DiI tissue-labeling paste consists of CM-DiI (C7000, C7001) mixed into an inert, water-resistant gel. The paste is ready to use as supplied and can be applied directly to live or fixed tissue specimens using the tip of a needle. This method of application improves the penetration of the dye into bundled neurons, labeling axons both on and below the surface.

DiIC18(5)-DS (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine-5,5'-Disulfonic Acid) (Invitrogen™)

The far red fluorescent, lipophilic carbocyanine DiIC18(5)-DS contains sulfonate groups to improve water solubility. It 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.

NeuroTrace™ Multicolor Tissue-Labeling Kit - DiO, DiI, DiD pastes, 500 mg each (Invitrogen™)

NeuroTrace® Multicolor Tissue-Labeling Kit contains 500 mg each of DiI, DiO or DiD mixed into an inert, water-resistant gel. The paste is ready to use as supplied and can be applied directly to live or fixed tissue specimens using the tip of a needle. This method of application improves the penetration of the dye into bundled neurons, labeling axons both on and below the surface.

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.

DiA; 4-Di-16-ASP (4-(4-(Dihexadecylamino)styryl)-N-Methylpyridinium Iodide) (Invitrogen™)

The lipophilic aminostyryl probe DiA inserts into membranes with its two alkyl tails and its fluorophore oriented parallel to the phospholipid acyl chain. When this dialkylaminostyryl probe binds to membranes, it exhibits a strong fluorescence enhancement; its fluorescence in water is minimal.

Vybrant™ CM-DiI Cell-Labeling Solution (Invitrogen™)

The Vybrant® CM-DiI cell-labeling solution is a dye delivery solution that can be added directly to normal culture media to uniformly label suspended or attached culture cells for use in cell-cell fusion, cellular adhesion and migration applications.

FM™ 1-43 Dye (N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) Pyridinium Dibromide) (Invitrogen™)

FM 1-43 membrane probe is an excellent reagent both for identifying actively firing neurons and for investigating the mechanisms of activity-dependent vesicle cycling. This water-soluble dye, which is nontoxic to cells and virtually nonfluorescent in aqueous medium, is believed to insert into the outer leaflet of the cell membrane where it becomes intensely fluorescent. In a neuron that is actively releasing neurotransmitters, the dye becomes internalized within the recycled synaptic vesicles and the nerve terminals become brightly stained. The nonspecific staining of cell-surface membranes can simply be washed off prior to viewing. FM 1-43 membrane probe is also available as 1 mg in a single vial (T-3163). FM 1-43FX membrane probe (F-35355), an analog of FM 1-43 membrane probe that can be fixed in place using aldehyde-based fixatives, is also available.

FAST DiO™ Solid; DiOΔ9,12-C18(3), ClO4 (3,3'-Dilinoleyloxacarbocyanine Perchlorate) (Invitrogen™)

FAST DiO is an unsaturated analog of DiO (DiOC18(3)) that reportedly migrates ~50% faster. It is weakly fluorescent in water but highly fluorescent and quite photostable when incorporated into membranes. 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™ Calcein Violet 450 AM Viability Dye (Invitrogen™)

Calcein Violet 450 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 Violet fluorescent dye. Apoptotic and dead cells with compromised cell membranes do not retain Calcein Violet 450. Calcein Violet 450 is optimally excited at 408 nm and has a peak emission of 450 nm. For fluorescent microscopy, Calcein Violet 450 can be detected using the appropriate filter sets. For flow cytometric analysis, it can be excited off of the violet laser line (405 nm) and detected using filters for Pacific Blue™/eFluor™ 450 (450/50). Co-staining with Annexin V or 7-AAD is recommended to allow the greatest resolution between live and dead/apoptotic cells.

Molecular weight: 600
Peak excitation: 408 nm
Peak emission: 450 nm

Calcein Violet 450 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.

Reported Application
Flow Cytometric Analysis, Microscopy

Hydroxystilbamidine, methanesulfonate (Invitrogen™)

Hydroxystilbamidine exhibits distinctively different fluorescence emission profiles when bound to DNA compared to RNA. This cationic dye is also frequently used as a retrograde neuronal tracer. Please note that the product offered prior to July 2001 under catalog number H-7599 was actually aminostilbamidine and not hydroxystilbamidine. Aminostilbamidine is now offered under a new catalog number, A-22850.

eBioscience™ Fura-2 AM Dye (Invitrogen™)

Fura-2 is a calcium imaging dye that binds to free Ca2+. Fura-2 AM is the cell-permeable acetoxymethyl (AM) ester form of Fura-2. Fura-2 is the preferred dye for ratiometric imaging microscopy with digital image analysis, especially when the alteration of excitation wavelengths is more practical than the detection of multiple emission wavelengths. Upon binding Ca2+, the excitation spectrum of Fura-2 shifts to shorter wavelengths between 300 and 400 nm, while the peak emission remains steady around 510 nm. The Kd of Fura-2 is highly dependent on pH, temperature, ionic strength and viscosity of the cytosol, thus great care should be taken when non-standard conditions are used.

Molecular weight: 1001.86
Peak excitation: variable depending on the concentration of free Ca2+, between 300 and 400 nm
Peak emission: 510 nm

Fura-2 AM should be reconstituted in high-quality, freshly opened DMSO. Once reconstituted, it should be protected from light and stored at -20°C. Avoid freeze-thawing.

Reported Application
Microscopy, Cell Labeling

Vybrant™ Multicolor Cell-Labeling Kit (DiO, DiI, DiD Solutions, 1 mL each) (Invitrogen™)

The Vybrant® Multicolor Cell Labeling Kit contains 1 mL each of the DiD, DiO and DiI cell-labeling solutions. These dye delivery solutions can be added directly to normal culture media to uniformly label suspended or attached culture cells for use in cell-cell fusion, cellular adhesion and migration applications.

eBioscience™ Cell Proliferation Dye eFluor™ 670 (Invitrogen™)

Cell Proliferation Dye eFluor™ 670 is a red fluorescent dye that can be used to monitor individual cell divisions. This fluorescent dye binds to any cellular protein containing primary amines, and as cells divide, the dye is distributed equally between daughter cells that can be measured as successive halving of the fluorescence intensity of the dye. Up to 6 generations may be visualized. Cell Proliferation Dye eFluor™ 670 can also be used for long term tracking of labeled cells. Analysis using two-parameter plots may provide better resolution of each generation, especially between undivided cells and the first generation. Cells labeled with Cell Proliferation Dye eFluor™ 670 may be fixed and permeabilized for analysis of intracellular targets using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers, such as the Foxp3 Transcription Factor Staining Buffer Set (cat. 00-5523) or the IC Fixation Buffer (cat. 00-8222) and Permeabilization Buffer (10X) (cat. 00-8333).

Cell Proliferation Dye eFluor™ 670 has a peak excitation of 647 nm and can be excited by the red (633 nm) laser line. It has a peak emission of 670 nm and can be detected with a 660/20 band pass filter (equivanet to APC, Alexa Fluor™ 647, or eFluor™ 660), making it compatible with applications that utilize GFP.

Cell Proliferation Dye eFluor™ 670 has a molecular weight of 792.6 and is supplied as a lyophilized powder. Each vial may be reconstituted to a stock concentration of 5 mM with 126 µL of anhydrous DMSO; once reconstituted it should be protected from light and stored at -20°C with dessicant. It is recommended to use the reconstituted dye within 6 months and to avoid freeze-thawing.

Reported Application
Flow Cytometric Analysis, Microscopy

eBioscience™ Fixable Viability Dye eFluor™ 520 (Invitrogen™)

Fixable Viability Dye eFluor™ 520 is a viability dye that can be used to irreversibly label dead cells prior to cryopreservation, fixation and/or permeabilization procedures. Unlike 7-AAD and propidium iodide, cells labeled with Fixable Viability Dyes can be washed, fixed, permabilized, and stained for intracellular antigens without any loss of staining intensity of the dead cells. Thus, using Fixable Viability Dyes allows dead cells to be excluded from analysis when intracellular targets are being studied. Fixable Viability Dyes may be used to label cells from all species.

Fixable Viability Dye eFluor™ 520 can be excited by the blue laser line (488 nm) and has a peak emission of 522 nm that can be detected using a 530/30 band pass filter (equivalent to FITC). Please make sure that your instrument is capable of detecting this dye. For compensation, it is recommended to use a sample of the cells of interest stained with the Fixable Viability Dye. If the percentage of dead cells is expected to be less than 5%, then it is recommended to take a small aliquot of cells and heat them at 65°C for 1 minute then immediately place on ice for 1 minute. After this treatment, the heat-killed cells can be combined 1:1 with live cells and then stained with the Fixable Viability Dye.

Fixable Viability Dye eFluor™ 520 is supplied as a pre-diluted solution prepared in high-quality, anhydrous DMSO. It should be protected from light and moisture. Store at less than or equal to -70°C with dessicant. It may be freeze-thawed up to 20 times. Allow vial to equilibrate to room temperature before opening.

Reported Application
Flow Cytometric Analysis