Fluorescent Cellular Stains...Neuro-DiI is derivative of orange-fluorescent DiI with structural...

Fluorescent Cellular Stains

Membrane and Membrane Potential Dyes

CellBrite™ Cytoplasmic Membrane Stains and Other Lipophilic Carbocyanine Dyes ........ p. 2Phospholipid Membrane Dyes ..... p. 3Membrane Potential Dyes .... p. 3Synaptic Vesicle Dyes ..... p. 4

Cytosolic Tracers ...... p. 5

Organelle Stains

MitoView™ Mitochondrial Dyes ....... p. 6LysoView™ Lysosome Stains ....... p. 7Nuclear Stains ...... p. 8

Fluorescent Lectins, Toxins, and Other Conjugates ..... p. 9

Apoptosis & Viability Stains ....... pp. 10-11

2

Fluorescent Membrane and Membrane Potential Dyes

Catalog No. Product Ex/Em Unit Size

30024 CellBrite™ Blue Cytoplasmic Membrane Labeling Kit 366/441 nm 50 assays

30021 CellBrite™ Green Cytoplasmic Membrane Labeling Dye 484/501 nm 1 mL

30022 CellBrite™ Orange Cytoplasmic Membrane Labeling Dye 549/565 nm 1 mL

30023 CellBrite™ Red Cytoplasmic Membrane Labeling Dye 644/665 nm 1 mL

30070 CellBrite™ NIR680 Cytoplasmic Membrane Labeling Dye (2 mM) 683/724 nm 100 uL





60011 DiO 484/501 nm 50 mg

60015 Neuro-DiO 484/501 nm 25 mg

60019 Neuro-DiO in vegetable oil 484/501 nm 200 uL

60035 Dilinoleyl DiO (FAST DiO™) 484/499 nm 5 mg

60012 DiOC14(3), hydroxyethanesulfonate 484/501 nm 50 mg

60010 DiI 549/565 nm 50 mg

60018 DiI in vegetable oil 549/565 nm 0.5 mL

60034 Dilinoleyl DiI (FAST DiI™) 549/565 nm 5 mg

60016 Neuro-DiI 549/565 nm 25 mg

60014 DiD 644/655 nm 50 mg

60017 DiR 748/780 nm 25 mg

Other Lipophilic Carbocyanine DyesCellBrite™ Cytoplasmic Membrane Dyes

Fast DiI and Fast DiO are trademarks of Thermo Fisher Scientific

CellBrite™ Cytoplasmic Membrane Dyes

Lipophilic carbocyanine dyes are widely used for labeling neurons in tissues by retrograde labeling, and to label membranes in a wide variety of cell types. The dyes are weakly fluorescent in aqueous phase, but become highly fluorescent in lipid bilayers. Staining is highly stable with low toxicity and very little dye transfer in between cells, making the dyes suitable for long-term cell labeling and tracking studies. Cell populations can be labeled with different fluorescent colors for identification after mixing. Double labeling can identify cells that have fused or formed stable clusters.

CellBrite™ Cytoplasmic Membrane Labeling Dyes are dye delivery solutions that can be added directly to normal culture media to label suspended or adherent cells in culture. We offer a selection of dyes with fluorescence ranging from blue to near-infrared. The CellBrite™ Blue Cytoplasmic Membrane Labeling Kit features DiB, a unique blue carbocyanine dye, and a cell loading agent. CellBrite™ Green (NeuroDiO), CellBrite™ Orange (DiI), CellBrite™ Red (DiD), and CellBrite™ NIR dyes are supplied as ready-to-add dye solutions. The near-infrared CellBrite™ NIR dyes have long emission wavelengths suitable for small animal imaging.

Other Lipophilic Carbocyanine Dyes for Membrane LabelingBiotium also offers a selection of stand-alone carbocyanine dyes. DiOC14(3) is derivative of green fluorescent DiO that is more soluble in aqueous media. Neuro-DiO has improved solubility in membranes, less tendency to form non-fluorescent aggregates, and better photostability compared to DiO. Neuro-DiI is derivative of orange-fluorescent DiI with structural features to make the probe diffuse faster in cell membranes. Neuro-DiO and Neuro-DiI are available in vegetable oil, a formulation commonly used for microinjection. The dilinoleyl carbocyanines (Fast DiI™ and Fast DiO™) have much faster lateral diffusion rate than regular carbocyanine dyes and thus are excellent for labeling neurons in tissues. Near-infrared DiR can be used for small animal imaging.

250 350 450 550 650 750 850Wavelength (nm)

CellBrite BlueCellBrite GreenCellBrite OrangeCellBrite RedCellBrite NIR680CellBrite NIR750CellBrite NIR770CellBrite NIR790

Abs

orba

nce

375 475 575 675 775 875Wavelength (nm)

CellBrite BlueCellBrite GreenCellBrite OrangeCellBrite RedCellBrite NIR680CellBrite NIR750CellBrite NIR770CellBrite NIR790E

mis

sion

Figure 2. Absorption (A) and emission (B) spectra of lipophilic carbocyanine dyes. CellBrite Blue and DiB have identical spectra. DiO, DiOC14(3), and Dilinoleyl DiO are spectrally similar to CellBrite Green (Neuro-DiO). Neuro-DiI, and Dilinoleyl DiI are spectrally similar to CellBrite Orange (DiI). CellBrite Red and DiD have identical spectra. DiR is spectrally similar to CellBrite NIR750.

Absorption spectra of CellBrite™ Cytoplasmic Membrane Dyes Fluorescence emission spectra of CellBrite™ Cytoplasmic Membrane Dyes

Figure 1. HeLa cells stained with 1 uM CellBrite NIR680 (magenta) for 20 minutes at 37oC. Nuclei were counterstained with Hoechst (blue).

A B

3


Phospholipid Membrane ProbesThese membrane probes are derived from natural phospholipids by modifying the head group with a fluorescent dye or biotin. The probes are useful for studies of vesicle trafficking, membrane fusion, and interaction of labeled membranes with other biomolecules. Red fluorescent phospholipids like TRITC-, Rhodamine-, and Texas Red®-DHPE have been used as fluorescence acceptors in combination with NBD-DHPE in FRET-based membrane-fusion assays.

Fast-Responding Membrane Potential DyesFast-responding membrane potential dyes are styryl dyes that undergo changes in fluorescence intensity in response to changes in membrane potential, on the order of 2-10% change in fluorescence per 100 mV. However, the dyes also undergo spectral shift with changes in membrane potential, allowing ratiometric measurements. Fast response dyes have been used to measure electrical activity in neural and cardiac cells.

Di-4-ANEPPS and Di-8-ANEPPS show changes in fluorescence excitation intensity that correlate with changes in membrane potential. Di-8-ANNEPPS is more hydrophobic and better retained in the outer leaflet of the plasma membrane than Di-4-ANNEPS, and therefore is more suitable for long-term membrane potential studies. It is also more photostable and less phototoxic than Di-4-ANNEPS.

Di-2-ANEPEQ (also known as JPW 1114) is a highly water soluble fast-responding dye that is usually introduced into cells by microinjection. Di-8-ANEPQ and Di-12-ANEPQ are successively more hydrophobic, and have been used for potential-sensitive retrograde labeling of neurons.

RH237, RH414, RH421, and RH795 are fast-responding potentiometric probes generally used for functional imaging of neurons. RH421 exhibits >20% fluorescence change per 100 mV on neuroblastoma cells. These dyes can differ in their physiological effects, for example RH414 causes arterial constriction during cortex staining, while the spectrally similar dye RH795 does not.

Translational Membrane Potential DyesTranslational (or slow-responding) membrane potential dyes undergo a change in their membrane distribution as a result of changes in membrane potential.

DiBAC4(3) is a translational membrane potential dye that redistributes within the cell membrane when membrane potential changes. The fluorescence of the dye is enhanced with membrane depolarization. The rate of fluorescence response of the dye is slower than styryl dyes like the ANEPPS dyes, but the fluorescence signal change is significantly larger.

DiOC2(3) has been used for measuring membrane potential in bacteria. The green fluorescent dye forms red fluorescent aggregates with increasing membrane potential, allowing ratiometric potential measurements.

DiOC5(3) and DiOC6(3) are two of the most widely used carbocyanine dyes for membrane potential measurements.

Tetramethylrhodamine ethyl ester (TMRE) and Tetramethylrhodamine methyl ester (TMRM) can be used for quantitative measurements of membrane potentials using the Nernst equation. The dyes do not form aggregates in cell membranes and have minimal interaction with membrane proteins. Thus, the transmembrane distribution of the dyes is directly related to membrane potential. The dyes are also used as membrane potential-dependent mitochondrial stains.

350 450 550 650 750

Em

ission

Abs

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Wavelength (nm)

Figure 1. Absorption and emission spectra of Di-4-ANEPPS and Di-8-ANEPPS bound to liposomes. The spectra of Di-2-ANEPEQ, Di-8-ANEPPQ, and Di-12-ANEPPQ are expected to be similar but red-shifted by 10-20 nm. In general, the spectra of styryl dyes undergo a large blue shift in going from organic solvent to cell membranes. The spectra shown here should be representative of the spectra in cell membranes for ANEPPS and ANEPEQ dyes.


60022 Biotin-DHPE N/A 10 mg

60023 Biotin-X-DHPE N/A 5 mg

60024 Fluorescein-DHPE 496/519 nm 5 mg

60025 NBD-PE 463/536 nm 10 mg

60028 TRITC-DHPE 540/566 nm 1 mg

60026 Rhodamine-DHPE 560/581 nm 5 mg

60027 Texas Red®-DHPE 582/601 nm 1 mg

Catalog No. Product Ex/Em

(MeOH) Unit Size

61011 DiBAC4(3) 493/516 nm 25 mg

70008 DiOC2(3) 482/497 nm 100 mg

70007 DiOC5(3) 482/497 nm 100 mg

70009 DiOC6(3) 484/501 nm 100 mg

70016 TMRE 549/574 nm 25 mg

70017 TMRM 548/573 nm 25 mg

Catalog No. Product Ex/Em

(MeOH)* Unit Size

61010 Di-4-ANEPPS 496/705 nm 5 mg

61012 Di-8-ANEPPS 498/713 nm 5 mg

61013 Di-2-ANEPEQ (JPW 1114) see Fig. 1 5 mg

61014 Di-8-ANEPPQ see Fig. 1 5 mg

61015 Di-12-ANEPPQ see Fig. 1 5 mg

61018 RH237 528/782 nm 5 mg

61016 RH414 532/706 nm 5 mg

61017 RH421 515/704 nm 25 mg

61019 RH795 530/712 nm 5 mg*Ex/Em is shown for methanol solution of dye. In cell membranes, spectra of styryl dyes are typically blue shifted by as much as 20 nm for absorption/excitation and 80 nm for emission (see Fig. 1).Texas Red is a registered trademark of Thermo Fisher Scientific

4


SynaptoGreen™ and SynaptoRed™ Nerve Terminal Dyes Nerve terminal probes were originally called FM® dyes. Now they are available from Biotium under the trademark names SynaptoGreen™ and SynaptoRed™, depending on their fluorescence emission. They are a series of fluorescent cationic styryl dyes developed to follow synaptic activity at neuromuscular junctions or synapses. The dyes label synaptic vesicles in neuronal tissues (Fig. 1) and cultured neurons (page 5, Fig. 1) in an activity-depending fashion. They also can be used to label endocytic vesicles in other cell types. These dyes have a lipophilic tail at one end and a highly hydrophilic, cationically charged head group at the other end as illustrated by the general structure below, where m is the number of carbons in the lipophilic tail and n is the number of double bonds linking the two aromatic rings in the dye.

Nerve terminal dyes are virtually non-fluorescent in aqueous solution, but become intensely fluorescent in membranes. Following nerve stimulation, the dye molecules are internalized in newly formed endocytic vesicles. During exocytosis, the dyes are released from the vesicles along with neurotransmitters, causing a decrease in fluorescence signal. As a result, the change in fluorescent intensity reflects the amount of endocytosis/exocytosis or synaptic activity. The rate of fluorescence increase during endocytosis, the “on-rate”, and the rate of fluorescence decrease during exocytosis, the “off-rate”, vary from dye to dye. In general, dyes with longer lipophilic tails and more double bonds have a higher affinity toward membrane and thus a higher on-rate and lower off-rate. Some styryl dyes can enter cells through ion channels; SynaptoGreen C18 and AM3-25 are high molecular weight dyes that cannot pass through ion channels, and have been used as controls to distinguish mechanisms of dye uptake.

Fixable Synaptic Vesicle DyesAM dyes and HM dyes are fixable nerve terminal dyes. After staining with these dyes, cells can be fixed and permeabilized for subsequent immunostaining. The AM dyes have an aldehyde-fixable amino group attached to the positively-charged head group of the dyes. HM1-43 is similar except that the amino group is replaced by a hydrazide, which is more reactive with aldehyde fixatives. AM dyes are more water-soluble (and therefore have a lower on-rate and higher off-rate) than the corresponding FM dye counterparts, while HM1-43 is more lipophilic than AM1-43 due to its neutral hydrazide group.

Background Quenchers and Nerve Terminal Staining KitsA common problem encountered with nerve terminal dyes is background fluorescence due to residual membrane staining, even after extensive washing. To reduce this background fluorescence, we offer three quencher or dye-clearing agents. ADVASEP-7, a sulfonated β-cyclodextrin, forms a water soluble inclusion complex with SynaptoGreen C4 that can be removed more effectively by washing. Biotium’s unique quencher, SCAS, reduces background fluorescence as soon as it is added to the preparation without the need for washing. Sulforhodamine 101 quenches SynaptoGreen background staining via fluorescent resonance energy transfer (FRET). We offer these reagents as individual products and in kits with dyes and the quencher/dye-clearing agents.

Figure 1. General structure of SynaptoGreen and SynaptoRed dyes, where m = 0-17 and n = 1 or 3.

Catalog No. Product Kit Components

70030 Nerve Terminal Staining Kit I 5 x 1 mg SynaptoGreen™ C4 (70022)250 mg ADVASEP-7 (70029)

70031 Nerve Terminal Staining Kit II (A) 1 mg AM1-43 (70024)100 mg ADVASEP-7 (70029)

70031-1 Nerve Terminal Staining Kit II (B) 1 mg of AM1-43 (70024) 100 mg SCAS (70037)

70032 Nerve Terminal Staining Kit III 5 x 1 mg SynaptoGreen™ C4 (70022)100 mg Sulforhodamine 101 (80101)

70034 Nerve Terminal Staining Kit V 5 x 1 mg SynaptoRed™C2 (70027)250 mg ADVASEP-7 (70029)

Catalog no. Size Product m* n* Ex/Em in

membranes Fixable?

70042 5 mgSynaptoGreen™ C1 0 1 ~480/600 nm No

70043 5 x 1 mg

70044 5 mg SynaptoGreen™ C2 (equivalent to FM®2-10) 1 1 ~480/600 nm No

70045 5 x 1 mg

70023 5 mgSynaptoGreen™ C3 2 1 ~480/600 nm No

70026 5 x 1 mg


70022 5 x 1 mg


70047 5 x 1 mg

70048 5 mg SynaptoGreen™ C18(equivalent to FM®3-25) 17 1 ~480/600 nm No

70049 5 x 1 mg

70024 1 mg AM1-43 3 1 ~480/600 nm Yes

70038 1 mg AM1-44 4 1 ~480/600 nm Yes

70036 1 mg AM2-10 1 1 ~480/600 nm Yes

70051 1 mg AM3-25 17 1 ~480/600 nm Yes

70053 1 mg HM1-43 3 1 ~480/600 nm Yes

70040 5 mgSynaptoRed™ C1 0 3 ~510/750 nm No

70041 5 x 1 mg

70021 5 mg SynaptoRed™ C2(equivalent to FM®4-64) 1 3 ~510/750 nm No

70027 5 x 1 mg

70019 5 mg SynaptoRed™C2M**(equivalent to FM®5-95) 1 3 ~510/750 nm No

70028 5 x 1 mg

70025 1 mg AM4-64 1 3 ~510/750 nm Yes

70039 1 mg AM4-65 3 3 ~510/750 nm Yes

70050 1 mg AM4-66 4 3 ~510/750 nm Yes

*See Figure 1 for general dye structures. **The positively-charged end of SynaptoRed C2M is a trimethylammonium group. FM is a registered trademark of Thermo Fisher Scientific.

Figure 1. The apical half of a single mouse vibrissal follicle, labeled with AM1-43. Image courtesy of Dr. David Corey, Harvard Medical School.

Nerve Terminal Staining Kits

5


90055 Biocytin N/A 100 mg

90060 Biocytin hydrazide N/A 25 mg

90057 Biotin ethylenediamine, hydrobromide (equivalent to Neurobiotin™) N/A 25 mg

90075 Biotin ethylenediamine, hydrochloride (functionally equivalent to Neurobiotin™) N/A 25 mg

80019 Fluorescein-biotin 494/518 nm 5 mg

90062 Biotin-4-fluorescein 494/523 nm 10 mg

80022 Biotin-rhodamine 110 502/524 nm 5 mg

80017 Lucifer Yellow Cadaverine Biotin-X, dipotassium salt 428/532 nm 10 mg

80104 DBA/Terbium for membrane fusion assay 276/490, 545 nm* 1 set

80105 SDIP/Europium for membrane fusion assay 250-320/~610 nm* 1 set

80012 DPX N/A 500 mg

90010 ANTS 353/520 nm 500 mg

Cytosolic Tracers

CF™ Dye HydrazidesWe offer a wide selection of our bright and photostable CF™ dyes in hydrazide form. Water-soluble and aldehyde-fixable, hydrazides can be microinjected as neuronal morphology tracers (Figure 1). See page 9 for a list of CF™ dye hydrazides.

Calcein and Calcein AMCalcein is a water soluble fluorescein derivative widely used for the study of cell membrane integrity that can be introduced by microinjection. Calcein-AM is a membrane-permeant AM ester of calcein that can be loaded into cells by incubation, where it is cleaved by esterases to release the dye. Calcein AM also can be used to quantitate cell viability, because dead cells will not hydrolyze the ester or retain the hydrolyzed dye (see page 10).

Other Fluorescent TracersLucifer Yellow CH and Lucifer Yellow cadaverine are aldehyde-fixable green fluorescent cytosolic tracers.

Hydroxystilbamidine (equivalent to Fluoro-Gold™) is a UV-excitable green fluorescent dye that has been used extensively as a retrograde tracer for neurons and as a histochemical stain. It is available in solid form and as a ready-to-use 4% solution.

True Blue is a UV-excitable retrograde polar tracer with blue fluorescence.

Sulforhodamines are highly water soluble red fluorescent dyes that can be used as polar tracers for neuronal morphology and intercellular junctions. Sulforhodamine B also has been used as a fixed cell protein stain for colorimetric quantitation of cell number.

Biotin DerivativesBiocytin (e-biotinoyl-L-lysine) is a cellular tracer that can be introduced by microinjection; biocytin-hydrazide is an aldehyde-fixable analog. Biotin ethylenediamine (equivalent to Neurobiotin™) is used as an anterograde and transneuronal tracer. Fluorescein-biotin, biotin-4-fluorescein, biotin-rhodamine 110, and Lucifer Yellow cadaverine biotin-X are fluorescent biotin derivatives for cell tracing or detection of biotin binding sites.

We also offer fluorescent biotin conjugates of CF™ dyes with superior brightness and photostability. See page 9.


80013 Calcein (high purity) 494/517 nm 100 mg

30026 Calcein AM Cell Viability Assay Kit

494/517 nm*

1000 assays

80011 Calcein AM 1 mg

80011-1 Calcein AM, 4 mM in anhydrous DMSO 100 uL

80011-2 Calcein AM, 1 mg/mL in anhydrous DMSO 1 mL

80011-3 Calcein AM 20 x 50 ug

80015 Lucifer Yellow CH, lithium salt

428/536 nm

25 mg

80016 Lucifer Yellow CH, potassium salt 25 mg

80018 Lucifer Yellow Cadaverine 25 mg

80017 Lucifer Yellow Cadaverine Biotin-X, dipotassium salt 428/532 nm 10 mg

80014 Hydroxystilbamidine (equivalent to Fluoro-Gold™) 361/536 nm 10 mg

80023 Hydroxystilbamidine (equivalent to Fluoro-Gold™), 4% in H2O

361/536 nm 200 uL

80020 True Blue, chloride 375/403 nm 5 mg

80100 Sulforhodamine B 565/586 nm 5 g

80101 Sulforhodamine 101 586/605 nm 500 mg

80102 Sulforhodamine G 529/548 nm 5 g Neurobiotin is a trademark of Vector Laboratories. Fluoro-Gold is a trademark of Fluorochrome.

*For complex.

Reagents for membrane fusion assaysThe principle of DPA/Terbium vesicle fusion assays is based on the fact that contact of the chelator dipicolinic acid (DPA) with terbium (III) instantly forms a complex that is ~10,000 times more fluorescent than free Tb3+. In the assay, separate vesicle populations are loaded with DPA or terbium. Fusion of the two types of vesicles results in fluorescence at 490 and 545 nm. Each set includes 1 gram each of terbium trichloride and DPA.

Biotium developed SDIP/Europium as an alternative to DPA/Tb3+ for vesicle fusion assays. The combination of SDIP/Eu3+ generates intense red fluorescence at 610 nm, with brighter intensity compared to DPA/Tb3+. Each set includes 10 mg SDIP and 25 mg EuCl3.

DPX is a positively charged quencher that has been used with the fluorescent dye ANTS for studies of vesicle fusion or membrane permeability. The complex of ANTS-DPX has minimal fluorescence, but when it is diluted due to membrane fusion or leakage, the dye becomes increasingly fluorescent.

Figure 1. Cultured rat hippocampal neurons microinjected with CF647 hydrazide (red) and stained with SynaptoGreen C4 (green, synaptic vesicles). Image courtesy of Professor Guosong Liu, Tsinghua University, Beijing.

*Hydrolyzed product

6

Mitochondrial Dyes

MitoView™ DyesLoss of mitochondrial membrane potential is a hallmark for apoptosis. Biotium offers our MitoView™ Blue (Fig. 1) and MitoView™ 633 (Fig. 3) dyes for membrane potential-sensitive staining of mitochondria (Fig. 4-5). We also offer MitoView™ Green (Fig. 2), a membrane-potential independent mitochondrial dye that can be used to image mitochondria following mitochondrial depolarization, or after fixation.

JC-1 and Other Mitochondrial DyesIn healthy cells, JC-1 dye aggregates in mitochondria as a function of membrane potential, resulting in red fluorescence (Ex/Em 585/590 nm) 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 (Ex/Em 510/527 nm) 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 in a variety of fluorescent colors. Red fluorescent TMRM (tetramethylrhodamine methyl ester) and TMRE (tetramethylrhodamine ethyl ester) are preferred dyes for quantitative membrane potential measurements. DASPEI is a dye with red fluorescence emission that is used to stain mitochondria in live cells. DiIC1(5) is a near-infrared carbocyanine dye that has been used to detect loss of mitochondrial membrane potential in apoptotic cells. Nonyl acridine orange (NAO) is a green fluorescent mitochondrial dye with staining that is not membrane potential dependent.

0

500

1000

1500

2000

Control CCCP Staurosporine

Geo

met

ric m

ean

(FL4

-H)

Figure 5. Flow cytometry analysis of Jurkat cells treated with CCCP to depolarize the mitochondrial membrane or staurosporine to induce apoptosis, resulting in decreased MitoView 633 staining.

Figure 1. HeLa cells stained with MitoView Blue (cyan) and RedDot™1 far-red nuclear stain (magenta).

Figure 2. HeLa cells stained with MitoView Green. Figure 3. HeLa cells stained with MitoView 633.

Figure 4. HeLa cells stained with MitoView Blue after A) no treatment, B) overnight staurosporine treatment to induce apoptosis, or C) 15 minute CCCP treatment to depolarize mitochondria. MitoView Blue shows reduced staining in late apoptotic cells and cells with depolarized mitochondrial membrane potential.

A. Untreated B. Staurosporine C. CCCP

Catalog No. Product Ex/Em Potential-

dependent? Unit Size

70052 MitoView™ Blue 398/440 nm Yes 20 x 50 ug

70054 MitoView™ Green 490/523 nm No 20 x 50 ug

70055 MitoView™633 622/648 nm Yes 20 x 50 ug

30001 JC-1 Mitochondrial Membrane Potential Detection Kit

510/527 nm*585/590 nm** Yes 100 assays

70011 JC-1, chloride salt 510/527 nm*585/590 nm** Yes 5 mg

70014 JC-1, iodide salt 510/527 nm*585/590 nm** Yes 5 mg

70016 TMRE 548/573 nm Yes 25 mg

70005 TMRE, 2 mM in DMSO 548/573 nm Yes 0.5 mL

70017 TMRM 548/573 nm Yes 25 mg

70018 DASPEI 461/589 nm Yes 100 mg

70015 DiIC1(5) 638/658 nm Yes 100 mg

70012 Nonyl acridine orange 495/522 nm No 50 mg

*Cytosol **Mitochondria

7

Lysosome Stains

LysoView™ DyesLysoView™ dyes are fluorescent stains for imaging lysosome localization and morphology in live cells. LysoView™ dyes belong to a family of lysosomotropic dyes that contain weakly basic amines that accumulate in acidic organelles. LysoView™ dye fluorescence is also pH-sensitive (Figure 2), resulting in specific lysosomal staining without a wash step. Choose red fluorescent LysoView™ 540 or far-red fluorescent LysoView™ 633.

We also offer "Light-On" LysoView™ 555, a UV-activatable lysosome stain. In cells, the dye initially shows low fluorescence, but brief exposure to UV excitation from a mercury arc lamp activates bright red fluorescence localizing to lysosomes (Figure 4). Lysosomal fluorescence fades over the course of several minutes after UV exposure, but can be re-activated in the same cells multiple times by exposure to UV light. Therefore the dye provides a novel tool for UV-activated, reversible fluorescence imaging of lysosomes.

Figure 3. LysoView 633 compared to LysoTracker® Deep Red. Live HeLa cells were stained for 10 minutes at 37oC with 1X LysoView 633 or 50 nM LysoTracker® Deep Red (Life Technologies) in cell culture medium. Both dyes were imaged using Cy®5 excitation/emission settings with the same gain settings. LysoView 633 (A) shows more specific punctate lysosomal staining with less cytoplasmic staining compared to LysoTracker® Deep Red (B).

A. LysoView™ 633

LysoTracker is a registered trademark of Thermo Fisher Scientific; Cy Dye is a registered trademark of GE Healthcare.

Figure 1. Live HeLa cells stained with 1X LysoView 540 (red). Nuclei are stained with Hoechst 33342 (Biotium catalog no. 40046).

A. Before UV exposure B. After UV exposure

Figure 4. UV-activated lysosomal fluorescence with "Light-On" LysoView 555. HeLa cells were stained with 1 uM Light-on LysoView 555 for 15 minutes at 37oC, then imaged using a Zeiss LSM 700 confocal microscope using a 40X objective and imaging settings for Cy®3. A. Before UV exposure, fluorescence was not detectable. B. After five seconds of exposure to UV light from a short arc lamp, bright red fluorescence localized to lysosomes was observed.


70061-T LysoView™ 540, 1000X in DMSO 541/634 nm* 10 uL

70061 LysoView™ 540, 1000X in DMSO 541/634 nm* 50 uL

70058 LysoView™633 (1000X after reconstitution) 634/659 nm 10 x 100 uL

70060-T "Light-On" LysoView™ 555, 1 mM in DMSO 554/583 nm* 10 uL

70060 "Light-On" LysoView™ 555, 1 mM in DMSO 554/583 nm* 50 uL

B LysoTracker® Deep Red

*pH ≤ 5

550 600 650 700 750 800

Em

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Wavelength (nm)

pH 2pH 3pH 4.6pH 6pH 7pH 8pH 9pH 9.6pH 11.6

pH 6

pH 7

pH ≥ 8

pH ≤ 4.6

625 650 675 700 725

Em

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Wavelength (nm)

pH 2pH 3pH 4pH 5pH 6pH 7pH 8pH 9

pH ≤ 5

pH 6

pH 7

pH 8

pH 9

Figure 2. pH dependence of LysoView 540 (A) and LysoView 633 (B)fluorescence emission.

A

B

8

Nuclear Stains

Figure 1. Absorption and emission spectra of FITC (green), Cy3 (orange) and RedDot2 (with DNA) (red). RedDot1 and RedDot2 have similar spectra.


40060-T

RedDot™1 662/694 nm*

25 uL40060 250 uL

40060-1 1 mL

40061-T

RedDot™2 662/694 nm*

25 uL

40061 250 uL

40061-1 1 mL

40011 DAPI

358/461 nm*

10 mg

40009 DAPI, dilactate 10 mg

40043 DAPI, 10 mg/mL in H2O 1 mL

23002 EverBrite™ Mounting Medium with DAPI 10 mL

23004 EverBrite™ Hardset Mounting Medium with DAPI 10 mL

40044 Hoechst 33258, 10 mg/mL in H2O 10 mL

40045 Hoechst 33258, pentahydrate 100 mg

40046 Hoechst 33342, 10 mg/mL in H2O 10 mL

40047 Hoechst 33342, trihydrochloride trihydrate 100 mg

40016 Propidium iodide (PI)

535/617 nm**

100 mg

40017 Propidium iodide, 1 mg/mL in H2O 10 mL

40048 Propidium iodide buffer, 50 ug/mL in PBS 2 mL

40039 Acridine Orange, 10 mg/mL in H2O 500/525 nm (DNA)460/650 nm (RNA) 10 mL

40037 7-AAD 546/647 nm* 1 mg

40010 Ethidium Homodimer I528/617**

1 mg

40014 Ethidium Homodimer I, 2 mM in DMSO 0.5 mL

40050 Ethidium Homodimer III530/620**

1 mg

40051 Ethidium Homodimer III, 1 mM in DMSO 200 uL

RedDot™1 and RedDot™2 Far-Red Nuclear StainsRedDot™1 and RedDot™2 are two far-red nuclear counterstains. RedDot™ dyes have broad excitation centered around 660 nm, and can be excited by several common laser lines. Their far-red emission is well-separated from other fluorophores (Figure 1), making the dyes useful counterstains for multicolor imaging, and for use with instruments that are not compatible with blue fluorescent nuclear stains like DAPI.

RedDot™1 rapidly and specifically stains nuclei in live cells (see p. 10, Fig. 1), and can be used for cell cycle analysis by flow cytometry or for cell normalization for In Cell Western™. RedDot™2 is membrane impermeant and can be used to selectively stain dead cells, or as a nuclear counterstain for fixed cells (Figures 2, 3). RedDot2 shows better nuclear specificity in fixed cells than Draq7™, which requires a blocking step for nuclear-specific staining (Figure 2).

400 500 600 700 800

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Wavelength (nm)400 500 600 700 800

Em

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Figure 3. Fixed and permeabilized HeLa cells stained with rabbit anti-COX IV and CF488A goat anti-rabbit (mitochondria, green), and mouse anti-Golgin 97 and CF555 goat anti-mouse (Golgi, red). Actin filaments are stained with CF405 phalloidin (blue) and nuclei are stained with RedDot2 (magenta).

DAPI and HoechstDAPI and Hoechst are widely used blue fluorescent nuclear counterstains. They are minor groove-binding DNA dyes that are minimally fluorescent in solution, but have strong fluorescence enhancement upon binding DNA. Hoechst dyes are membrane permeant and can be used for live or fixed cell staining and cell cycle analysis. Hoechst 33342 is slightly more cell permeable than Hoechst 33258. DAPI is commonly used as a fixed cell counterstain. DAPI dilactate is the same as DAPI but with a different counterion that makes it more soluble in water. We also offer EverBrite™ Mounting Media with DAPI for combination antifade and counterstaining.

Other Nucleic Acid DyesPropidium iodide (PI) is a membrane impermeant red fluorescent DNA/RNA stain commonly used to selectively stain dead cells for flow cytometry and for cell cycle analysis.

Our Live-or-Dye NucFix™ Red is a fixable alternative to PI for dead cell nuclear staining for microscopy or flow cytometry (see page 11).

Acridine Orange (AO) is cell membrane permeant and stains double-stranded DNA green (525 nm emission) and RNA red (650 nm emission).

7-AAD is a 488 nm-excitable dye with far-red emission, useful for flow cytometry. It is membrane impermeant and can be used for selective staining of dead cells, or cell cycle analysis.

Ethidium homodimer I (EthD-I) is a red fluorescent (Ex/Em with DNA 528/617 nm) membrane impermeant dead cell stains that bind DNA and RNA. Biotium’s Ethidium homodimer III is spectrally similar, but stains nucleic acids 70% brighter than EthD-I.

Staining kits with a variety of dead cell nuclear stains and apoptosis probes are listed on page 10.

*With DNA**With DNA or RNA

Figure 2. Formaldehyde fixed and detergent permeabilized HeLa cells stained with 1X RedDot2 (A) or 3 uM Draq7™ (B) in PBS for 10 minutes. RedDot2 staining is highly selective for the nucleus, while Draq7™ stains the nucleus and cytoplasm unless a separate blocking step is performed. Actin is stained with CF488A phalloidin (green).

Draq7 is a trademark of Biostatus, Ltd. Cy Dye is a registered trademark of GE Healthcare. In Cell Western is a trademark of LI-COR, Inc.

A. RedDot™2 B Draq7™

9

CF™ Dye Lectins, Toxins, and Other Conjugates

CF™ Dye ConjugatesBiotium offers a wide selection of probes for cell staining conjugated to CF™ dyes, our line of next-generation fluorescent dyes. CF™ dyes have advantages in brightness, photostability, and water solubility compared to dyes like Alexa Fluor®, DyLight®, and Cy® dyes. See our CF™ dye selection guide at www.biotium.com for more information and side-by-side dye comparison data.

Conjugate Application

Biotin Cytoplasmic tracer (see p. 5); biotin binding site detection

a-Bungarotoxin (a-BTX) Acetylcholine receptor/neuromuscular junction probe

Bovine serum albumin (BSA) Fluid-phase endocytosis tracer; in vivo blood flow tracer

Cholera Toxin Subunit B GM1 receptor probe; lipid raft, endocytic vesicle, neuronal tracing

Concanavalin A (Con A) Lectin; binds α-D-mannosyl and α-D-glucosyl groups, stains yeast cell wall

Dextran, MW 10,000, anionic, fixable Fluid-phase endocytosis tracer

Hydrazide Fixable, water-soluble cytoplasmic tracer (see p. 5)

Phalloidin Filamentous actin probe

Peanut agglutinin (PNA) Lectin; specific for terminal b-galactose

Streptavidin Detection of biotinylated probes

Transferrin (human) Recycling endosome tracer

Wheat germ agglutinin (WGA) Lectin, binds N-acetyl-D-glucosamine and sialic acid; bacterial Gram stain, stains yeast bud scars

Figure 1. Neuromuscular junction in rat skeletal muscle stained with CF633 a-bungarotoxin (magenta). Nuclei are stained with DAPI (blue).

Dye Conjugate Ex/Em Biotin a-BTX BSA Cholera

Toxin B Con A Dextran Hydrazide Phalloidin PNA Streptavidin Transferrin WGA

CF™350 347/448 nm 29015 92151 00049 29031 29021

CF™405S 404/431 nm 00002 29032 29027

CF™405M 408/452 nm 00034 29033

CF™430 426/498 nm 00054

CF™440 440/515 nm 00055

CF™488A 490/515 nm 00005 20289 00070 29016 80110 92152 00042 29060 29034 00081 29022

CF™532 527/558 nm 00051 29030

CF™543 541/560 nm 00026 80111 00043 29043 00082

CF™555 555/565 nm 00018 80112 92153 00040 29038

CF™568 562/583 nm 80029 00006 00071 80113 92154 00044 29061 29035 00083

CF™594 593/614 nm 00007 20290 00072 29017 80114 92158 00045 29062 29036 00084 29023

CF™633 630/650 nm 80031 00009 29018 92156 00046 29037 29024

CF™640R 642/662 nm 80032 00004 20291 00073 29019 80115 92157 00050 29063 29041 00085 29026

CF™647 650/665 nm 92136 00041 29039

CF™660C 667/685 nm 00052

CF™660R 663/682 nm 00047 29040

CF™680 681/698 nm 20292

CF™680R 680/701 nm 00003 80116 00048 29042 00086 29025

Figure 2. S. cerevisiae yeast stained with CF488A WGA and CF594 ConA. ConA (red) stains the cell wall, while WGA (green) preferentially stains bud scars.

Don’t see what you’re looking for? We regularly add new CF™ dye products to our catalog according to customer demand. Be sure to check our website for updates. If you are looking for a CF™ dye product not listed in our catalog, contact us at [email protected]. We may be able to add it as a new product, or perform a custom conjugation for you.

Figure 3. HeLa cells stained with cholera toxin subunit B conjugated to CF488A (green). Nuclei are stained with DAPI (blue).

Alexa Fluor and DyLight are registered trademarks of Thermo Fisher Scientific. Cy Dye is a registered trademark of GE Healthcare.

10

Apoptosis and Viability Stains

Catalog No. Product Ex/Em Unit Size29012 CF™350 Annexin V, 50 ug/mL 347/448 nm 0.5 mL29009 CF™405M Annexin V, 50 ug/mL 408/452 nm 0.5 mL29005 CF™488A Annexin V, 50 ug/mL 490/515 nm 0.5 mL29004 CF™555 Annexin V, 50 ug/mL 555/565 nm 0.5 mL29010 CF™568 Annexin V, 50 ug/mL 562/583 nm 0.5 mL29011 CF™594 Annexin V, 50 ug/mL 593/614 nm 0.5 mL29008 CF™633 Annexin V, 50 ug/mL 630/650 nm 0.5 mL29014 CF™640R Annexin V, 50 ug/mL 642/662 nm 0.5 mL29003 CF™647 Annexin V, 50 ug/mL 650/665 nm 0.5 mL29069 CF™660R Annexin V, 50 ug/mL 663/682 nm 0.5 mL29007 CF™680 Annexin V, lyophilized 681/698 nm 25 ug29070 CF™680R Annexin V, lyophilized 680/701 nm 25 ug29006 CF™750 Annexin V, lyophilized 755/777 nm 25 ug29046 CF™770 Annexin V, lyophilized 770/797 nm 25 ug29047 CF™790 Annexin V, lyophilized 784/806 nm 25 ug

30060 CF™488A Annexin V and PI Apoptosis Assay Kit

490/515 nm, 535/617 nm 100 assays

30061 CF™488A Annexin V and 7-AAD Apoptosis Assay Kit

490/515 nm, 546/647 nm 100 assays

30065 Apoptosis and Necrosis Assay Plus 490/515 nm, 530/620 nm 50 assays

30066 Apoptotic, Necrotic and Healthy Cells Assay Plus

358/461 nm, 490/515 nm, 530/620 nm

50 assays

30026 Calcein AM Cell Viability Assay Kit 494/517 nm 1000 assays

30002 Viability/Cytotoxity Assay Kit for Animal Live & Dead Cells

494/517 nm, 530/620 nm 300 assays

NucView™ Caspase-3 SubstratesNucView™ Caspase-3 Substrates are cleaved by caspases to stain the nuclei of apoptotic cells with fluorescence. Unlike FLICA substrates, NucView substrates do not inhibit caspase activity, allowing caspase-3/7 activity to be monitored in individual intact cells in real time.

Green fluorogenic NucView™ 488 Caspase-3 Substrate (Ex/Em 504/534 nm) has been validated in more than a hundred published studies and cell types, and hs been validated in real-time kinetic studies using the IncuCyte® Live Cell Analysis System (Essen Bioscience). We also offer blue fluorogenic NucView™ 405 Caspase-3 Substrate (429/469 nm) and orange fluorogenic NucView™ 530 Caspase-3 Substrate (528/563 nm) for multi-color flexibility.

CF™ Dye Annexin V Conjugates and Cell Viability Kits

NucView™ Caspase-3 Substrates and KitsCatalog

No. Product Ex/Em Unit Size

10402-T NucView™ 488 Caspase-3 Substrate, 1 mM in DMSO 504/534 nm* 10 uL

10402 100 uL10403-T NucView™ 488 Caspase-3 Substrate,

1 mM in PBS 504/534 nm* 10 uL10403 100 uL

10405-T NucView™ 405 Caspase-3 Substrate, 1 mM in DMSO 429/469 nm* 10 uL

10405 100 uL10406-T NucView™ 530 Caspase-3 Substrate,

1 mM in DMSO 528/563 nm* 10 uL10406 100 uL

30067 Dual Apoptosis Assay with NucView™ 488 Caspase-3 Substrate and CF™594 Annexin V

504/534 nm, 593/614 nm 50 assays

30073 Dual Apoptosis Assay with NucView™ 488 Caspase-3 Substrate and CF™640R Annexin V

504/534 nm, 642/662 nm 50 assays

30062 NucView™ 488 and MitoView™ 633 Apoptosis Kit

504/534 nm, 622/648 nm 100 assays

30072 NucView™ 488 and RedDot™2 Apoptosis & Necrosis Kit

504/534 nm, 662/694 nm 100 assays

Figure 3. Apoptotic Jurkat cells stained with NucView 488 Caspase-3 Substrate (green) and CF594 Annexin V (red).

CF™ Dye Annexin V ConjugatesFluorescent conjugates of Annexin V can be used to stain phosphatidylserine on the surface of apoptotic cells. We offer Annexin V conjugates of our exceptionally bright and photostable CF™ dyes for flow cytometry or fluorescence microscopy. Our CF™488A and CF™594 Annexin V conjugates have been validated in real-time kinetic imaging studies using the IncuCyte® Live Cell Analysis System (Essen Bioscience).

We also offer kits combining CF™488A Annexin V with red dead cell stains PI or 7-AAD (see p. 8). The Apoptosis and Necrosis Kit Plus includes CF™488A and EthD-III (see p. 8), while the Apoptotic, Necrotic and Healthy Cells Kit Plus also includes Hoechst to stain the total cell population.

Figure 1. HeLa cells stained with NucView 405 Caspase-3 Substrate (blue), CF488A Annexin V (green), and RedDot1 (red). Apoptotic cells show blue nuclear NucView staining and green membrane staining with Annexin V.

IncuCyte is a registered trademark of Essen Bioscience.

Figure 2. HeLa cells stained with the Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells. Healthy cells stain green after incubation with calcein-AM (see p. 5), while dead cells stain red with EthD-III (see p. 8).

*Cleavage product with DNA

11

Catalog No.50 reactions

Catalog No.200 reactions Product Ex/Em

32002-T 32002 Live-or-Dye™ 350/448 347/448 nm

32003-T 32003 Live-or-Dye™ 405/452 408/452 nm

32009-T 32009 Live-or-Dye™ 405/545 395/545 nm

32004-T 32004 Live-or-Dye™ 488/515 490/515 nm

32005-T 32005 Live-or-Dye™ 568/583 562/583 nm

32006-T 32006 Live-or-Dye™ 594/614 561/624 nm

32007-T 32007 Live-or-Dye™ 640/662 642/662 nm

32008-T 32008 Live-or-Dye™ 750/777 755/777 nm

32010-T 32010 Live-or-Dye NucFix™ Red 520/610 nm

Live-or-Dye™ Fixable Viability StainsBiotium offers a selection of eight different amine-reactive Live-or-Dye™ viability stains spanning the fluorescence spectrum, for maximal flexibility in multi-color analysis. The membrane-impermeant dyes selectively enter dead cells and covalently label intracellular proteins. They can be used to discriminate dead from live cells in flow cytometry or microscopy (Fig. 1). Live-or-Dye™ labeling is extremely stable, allowing the cells to be fixed and permeabilized without loss of fluorescence or dye transfer between cells.

Figure 2. Ethanol-treated HeLa cells stained with NucFix Red (red) and Hoechst (blue). Dead cells show bright red nuclear fluorescence. Identical staining is seen when cells are fixed and permeabilized after staining.

Live-or-Dye™ and Live-or-Dye NucFix™ Red Fixable Viability Stains

Apoptosis and Viability Stains

Live-or-Dye NucFix™ Red Viability StainLive-or-Dye NucFix™ Red is a unique, cell membrane-impermeant dye that specifically stains the nuclei of dead cells (Fig. 2). Unlike other nuclear viability stains such as propidium iodide (PI), NucFix™ labeling is extremely stable, allowing the cells to be fixed and permeabilized without loss of fluorescence or dye transfer between cells.

Figure 1. A. Discrimination of live and dead cells by flow cytometry using Live-or-Dye Fixable Viability Stains. Live or heat-killed Jurkat cells were stained with the Live-or-Dye cell stain shown on each histogram x-axis. Heat killed cells (solid peaks) showed much higher fluorescence intensity compared to live cells (white peaks), allowing the two populations to be clearly distinguished. Results are shown for unfixed cells; nearly identical histograms were observed after fixation with 2% formaldehyde followed by permeabilization with 0.1% Triton X-100. B. Discrimination of live and dead cells in fluorescence microscopy using Live-or-Dye 488/515. Ethanol-treated HeLa cells were stained with Live-or-Dye 488/515 cell stain. After staining cells were fixed with 4% formaldehyde followed by permeabilization with 0.1% Triton X-100, and then stained with Hoechst to label both live and dead cells. Killed cells show bright green fluorescent staining, compared to no staining seen in live cells (blue Hoechst-stained nuclei), allowing the two populations to be clearly distinguished.

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