Functional Dyes and Reagents

• Cell Tracking / Labeling
• Cell Proliferation and Division
• Cell Viability
• DNA Content, Cell Cycle Analysis and Identification of Nucleated Cells
• Calcium Flux
• Mitochondrial Membrane Potential

Cell Tracking / Labeling
Dye	Excitation	Emission	Function	Benefits	Limitations	Links
CFSE	494 nm	521 nm	Labels cytoplasmic proteins	• Good for short- & long-term labeling of cells (days – weeks) for flow cytometry or microscopy • Can be used to track cell division history (see Cell Proliferation Dyes)	• Labeling cells with too much dye may inhibit function & significantly increases compensation values (flow cytometry) • Cannot be used in combination with FITC, Alexa Fluor® 488, or GFP	65-0850
Cell Proliferation Dye eFluor® 670	647 nm	670 nm	Labels cellular proteins	• Good for short- & long-term labeling of cells (days – weeks) for flow cytometry or microscopy • Can be used to track cell division history (see Cell Proliferation Dyes) • Compatible with GFP	• Labeling cells with too much dye may inhibit function & significantly increases compensation values (flow cytometry) • Cannot be used in combination with APC or Alexa Fluor® 647	65-0840
CellVue® Jade	478 nm	508 nm	Labels cell membrane	• Good for short term labeling of cells (1-7 days) for flow cytometry or microscopy	• Labeling cells with too much dye or for too long will kill cells • Dye can be transferred to other cells through trogocytosis • Does not divide evenly between daughter cells for tracking cell division • Cannot be used in combination with FITC, Alexa Fluor® 488, or GFP	88-0876
CellVue® Lavender	420 nm	461 nm	Labels cell membrane	• Good for short term labeling of cells (1-2 days) for flow cytometry or microscopy	• The dimmest of the CellVue dyes • Labeling cells with too much dye or for too long will kill cells • Dye can be transferred to other cells through trogocytosis • Does not divide evenly between daughter cells for tracking cell division • Cannot be used in combination with eFluor® 450 or Pacific Blue®	88-0873
CellVue® Maroon	647 nm	667 nm	Labels cell membrane	• Good for short term labeling of cells (1-7 days) for flow cytometry or microscopy	• Labeling cells with too much dye or for too long will kill cells • Dye can be transferred to other cells through trogocytosis • Does not divide evenly between daughter cells for tracking cell division • May not be compatible with other 633 nm-excitable fluorochromes	88-0870
CellVue® Burgundy	683 nm	707 nm	Labels cell membrane	• Good for short term labeling of cells (1-7 days) for flow cytometry or microscopy	• Labeling cells with too much dye or for too long will kill cells • Dye can be transferred to other cells through trogocytosis • Does not divide evenly between daughter cells for tracking cell division • May not be compatible with other 633 nm-excitable fluorochromes	88-0872
CellVue® Plum	652 nm	671 nm	Labels cell membrane	• Good for short term labeling of cells (1-7 days) for flow cytometry or microscopy	• Labeling cells with too much dye or for too long will kill cells • Dye can be transferred to other cells through trogocytosis • Does not divide evenly between daughter cells for tracking cell division • May not be compatible with other 633 nm-excitable fluorochromes	88-0871
CellVue® NIR780	745 nm	776 nm	Labels cell membrane	• Good for short term labeling of cells (1-7 days) for flow cytometry (can be excited by the red laser line) or microscopy	• Labeling cells with too much dye or for too long will kill cells • Dye can be transferred to other cells through trogocytosis • Does not divide evenly between daughter cells for tracking cell division • Cannot be used in combination with APC-eFluor® 780 or APC-Alexa Fluor® 750	88-0875
CellVue® NIR815	786 nm	814 nm	Labels cell membrane	• Good for short term labeling of cells (1-7 days) for in vivo imaging	• Labeling cells with too much dye or for too long will kill cells • Dye can be transferred to other cells through trogocytosis	88-0874

Cell Proliferation / Cell Division
Dye	Excitation	Emission	Function	Benefits	Limitations	Links
CFSE	494 nm	521 nm	Labels cytoplasmic proteins	• Can identify up to 8 individual cell divisions as dye is divided equally between daughter cells	• Labeling cells with too much dye may inhibit function & significantly increases compensation values • Cannot be used in combination with FITC, Alexa Fluor® 488, or GFP	65-0850
Cell Proliferation Dye eFluor® 670	647 nm	660 nm	Labels cellular proteins	• Can identify up to 6 individual cell divisions as dye is divided equally between daughter cells • Compatible with GFP	• Labeling cells with too much dye may inhibit function & also significantly increases compensation values • Cannot be used in combination with APC or Alexa Fluor® 647	65-0840

Cell Viability Dyes
Dye	Excitation	Emission	Function	Benefits	Limitation	Links
Fixable Viability Dye eFluor® 450	405 nm	450 nm	Labels dead cells	• Permanently labels dead cells allowing them to be excluded from analysis even when the cell preparation is being analyzed by flow cytometry for intracellular targets	• Cannot be used in combination with Pacific Blue® or eFluor® 450	65-0863
Fixable Viability Dye eFluor® 660	633 nm	660 nm	Labels dead cells	• Permanently labels dead cells allowing them to be excluded from analysis even when the cell preparation is being analyzed by flow cytometry for intracellular targets	• Cannot be used in combination with APC or Alexa Fluor® 647	65-0864
Fixable Viability Dye eFluor® 780	633 nm	780 nm	Labels dead cells	• Permanently labels dead cells allowing them to be excluded from analysis even when the cell preparation is being analyzed by flow cytometry for intracellular targets	• Cannot be used in combination with APC-eFluor® 780 or APC-Alexa Fluor® 750	65-0865
Calcein AM	495 nm	515 nm	Labels live cells	• Can be used to identify live cells by flow cytometry • Non-fluorescent compound freely enters live cells and intracellular esterases converts it to a fluorescent dye • Measures intracellular esterase activity in live cells • Is not retained in dead cells with compromised cell membranes	• Will not be retained in cells after fixation or permeabilization and therefore, is not useful for intracellular staining protocols • High concentrations of dye will require greater compensation out of PE, PerCP-Cy5.5, and PE-Cy7 channels • May require co-staining with Annexin, 7-AAD, or propidium iodide to optimally separate live/dead cells • Cannot be used in combination with FITC, Alexa Fluor® 488, or GFP	65-0853
Calcein Violet AM	408 nm	450 nm	Labels live cells	• Can be used to identify live cells by flow cytometry • Excited by the violet laser line • Non-fluorescent compound freely enters live cells and intracellular esterases converts it to a fluorescent dye • Measures intracellular esterase activity in live cells • Is not retained in dead cells with compromised cell membranes	• Will not be retained in cells after fixation or permeabilization and therefore, is not useful for intracellular staining protocols • Cannot be used in combination with Pacific Blue® or eFluor® 450	65-0854
Calcein Blue AM	360 nm	445 nm	Labels live cells	• Can be used to identify live cells by flow cytometry • Excited by the UV laser line • Non-fluorescent compound freely enters live cells and intracellular esterases converts it to a fluorescent dye • Measures intracellular esterase activity in live cells • Is not retained in dead cells with compromised cell membranes	• Will not be retained in cells after fixation or permeabilization and therefore, is not useful for intracellular staining protocols	65-0855
Propidium Iodide	488 nm	617 nm	Labels dead cells	• Allows exclusion of dead cells from flow cytometric analysis • Enters cells with compromised cell membranes and binds dsDNA/RNA • Cannot pass through intact cell membranes • May also be used for DNA content/cell cycle analysis (see DNA content, Cell cycle analysis, Identification of nucleated cells)	• Will not be retained in cells after fixation or permeabilization and therefore, is not useful for intracellular staining protocols	00-6990
7-AAD	548 nm	647 nm	Labels dead cells	• Allows exclusion of dead cells from flow cytometric analysis • Enters cells with compromised cell membranes and binds dsDNA • Cannot pass through intact cell membranes • May also be used for DNA content/cell cycle analysis (see DNA content, Cell cycle analysis, Identification of nucleated cells)	• Will not be retained in cells after fixation or permeabilization and therefore, is not useful for intracellular staining protocols	00-6993

DNA Content, Cell Cycle Analysis, and Identification of Nucleated Cells
Dye	Excitation	Emission	Function	Benefits	Limitations	Links
Nuclear RED (DRAQ5™)	488-647 nm	>670 nm	Labels dsDNA	• Binds DNA stoichiometrically for analysis of cell cycle and ploidy • Does not require fixation/ permeabilization • Can be used as a nuclear counterstain for microscopy • Can be used to distinguish nucleated/nonnucleated cells for flow cytometry	• Broad excitation / emission spectra may limit use in multi-color flow cytometry & may not be compatible with PerCP-eFluor® 710/PerCP-Cy5.5, PE-Cy7, APC/Alexa Fluor® 647, Alexa Fluor® 700, or APC-eFluor® 780/APC-Alexa Fluor® 750 • For cell cycle analysis, emission should be collected with far-red filters (780/60) to obtain optimal CVs • For cell cycle analysis, it may be necessary to label cells for a longer period of time and with a higher concentration to obtain optimal CVs	65-0880
Nuclear ORANGE (CyTRAK Orange™)	488-550 nm	610 nm	Labels dsDNA	• Binds DNA • Does not require fixation/ permeabilization • Can be used to distinguish nucleated and nonnucleated cells for flow cytometry	• Background staining of the cytoplasm may limit its use as a nuclear counterstain in fluorescent microscopy	65-0881
Propidium Iodide	488 nm	617 nm	Labels dsDNA/RNA	• Binds DNA/RNA stoichiometrically for analysis of cell cycle and ploidy • May also be used for live/dead discrimination in flow cytometry	• Must fix/permeabilize cells • Requires degradation of cellular RNA for DNA/cell cycle analysis • Has a broad emission spectra making it difficult to use in multi-color flow cytometric applications	00-6990
7-AAD	548 nm	647 nm	Labels dsDNA	• Specifically binds DNA (does not bind to RNA) stoichiometrically for analysis of cell cycle and ploidy • May also be used for live/dead discrimination in flow cytometry	• Must fix/permeabilize cells	00-6993

Calcium Flux
Dye	Excitation	Emission	Function	Benefits	Limitations	Links
eFluor® 514 Calcium Sensor Dye	490 nm	514 nm	Fluorescence intensity increases upon binding free Ca2+	• Allows qualitative/semi-quantitative measurements of intracellular free Ca2+ • Compatible with flow cytometry, fluorescence microscopy, microplate readers, and spectrophotometry • Better cellular uptake and brighter than Fluo-3 or Fluo-4	• Only semi-quantitatve	65-0859
Indo-1 AM	346 nm	410 nm (bound) 485 nm (unbound)	Peak emission decreases upon binding free Ca2+	• Allows quantitative measurement of free Ca2+ • Compatible with flow cytometry, fluorescence microscopy, microplate readers, and spectrophotometry • Ratiometric measurements possible	• Compensation between bound/ unbound emission may be difficult • Not compatible with eFluor® 450, 605NC, 625NC, 650 NC, or other violet laser-excitable dyes	65-0856 65-0857
Fura-2 AM	300 nm (bound) 400 nm (unbound)	510 nm	Peak excitation decreases upon binding free Ca2+	• Ideal for measuring low to medium concentrations of Ca2+ by spectrophotometry or microscopy (when specific excitiation wavelengths can be used) • Compatible with ratiometric imaging microscopy	• Not for use with flow cytometry • Kd is highly sensitive to changes in pH, temperature, ionic strength and viscosity of the cytosol	65-0858

Mitochondrial Membrane Potential
Dye	Excitation	Emission	Function	Benefits	Limitations	Links
JC-1	515 nm	530 nm (monomer/ depolarized) 590 nm (aggregates/ polarized)	Measures polarization/ depolarization of mitochondrial membrane	• Selectively enters mitochondria & can be used to indicate the initiation of apoptosis • Changes in fluorescence is reversible • Compatible with flow cytometry and fluorescent spectrophotometry • Provides qualitative and quantitative measurements	• Compensation between the monomer and aggregate emission can be difficult	65-0851