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Let's move cell health forward together Sensitive indicators of cell health and stress Mitochondrial stains Mitochondria are prominent cellular markers, and mitochondrial function is a highly sensitive indicator of cell health and stress. Depolarization of the inner mitochondrial membrane potential is a reliable indicator of mitochondrial dysfunction, which has been increasingly implicated in drug toxicity. Life Technologies provides a range of Molecular Probes® tools for tracking mitochondrial location and investigating mitochondrial function using cell stains, functional probes, and fluorescent proteins. •Choice of technologies for optimized performance in each application •Wide range of colors for counterstaining with other fluorescent reporters •Functional probes for reactive oxygen species and membrane potential Fluorescent proteins: Mitochondrial behavior can be visualized dynamically in live cells using CellLight® technology. CellLight® reagents express fusion proteins with red or green fluorescence for imaging in live cells or after formalin fixation. Antibody conjugates: With Molecular Probes® antibody conjugates you can label mitochondria in fixed cells or frozen tissue sections even after paraffin embedding. (Unconjugated antibodies require the use of secondary detection labels.) MitoTracker® Probes: The MitoTracker® family of probes stains mitochondria in live cells through a variety of mechanisms. Some probes load according to mitochondrial membrane potential and some are retained after fixation. Functional probes: MitoSOX™ Red Mitochondrial Superoxide Indicator is an indicator of superoxide activity in mitochondria, and MitoHealth stain can serve as a marker for mitochondrial membrane potential in high content screening applications. For more information go to lifetechnologies.com/mitochondria For research use only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO07205 0 13 Cellular scaffold and the ultimate counterstain target Cytoskeleton stains The cytoskeleton has a role in organelle transport, cell division, motility, and signaling, making it central to normal growth processes and critical in disease models. Cytoskeleton stains enable research on the cellular scaffold, and they also serve as counterstains or fiducial markers for cellular orientation and co-localization in both manual and automated imaging systems. Molecular Probes® products are the most cited cytoskeleton stains and support any application. Alexa Fluor® conjugates, antibodies, and fluorescent proteins provide the leading technology solutions for labeling the major classes of cytoskeletal proteins. •Bright fluorescence for visualizing fibers and tubules •Choice of technologies for optimized performance in any application •Wide choice of colors for counterstaining with other fluorescent reporters Fluorescent proteins: Both actin and tubulin behavior can be visualized dynamically in live cells using CellLight® technology. CellLight® reagents express fusion proteins with red or green fluorescence for imaging in live cells or after formalin fixation. Antibody conjugates: With Molecular Probes® antibody conjugates you can label actin or tubulin in fixed cells or frozen tissue sections even after paraffin embedding. (Unconjugated antibodies require the use of secondary detection labels.) Phalloidin conjugates: The most common actin label for fixed and permeabilized cells is available conjugated to a range of high performance dyes. Brighter staining and resistance to photobleaching in a wide range of colors for multiplexing. For more information go to lifetechnologies.com/cytoskeleton For research use only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO07205 0113 TubulinTracker: This Taxol derivative stains polymerized tubulin for live cell imaging. Classic counterstains and cell death markers Nuclear stains The nucleus is routinely stained to serve as a fiducial marker in automated imaging systems. In addition, nucleic acid stains that show enhanced fluorescence on binding to DNA are frequently used as markers of membrane permeability and cell death. •Choice of technologies for optimized performance in each application •Wide range of colors for counterstaining with other fluorescent reporters •Applications from dead-cell assays to counterstaining Counterstains: Nuclear counterstains with blue fluorescence like Hoechst 33342 and DAPI provide spatial information about the cell and leave popular spectral channels open for multiplexing. SYTO® dyes stain DNA and RNA in live or fixed cells. They serve as counterstains in a range of colors from green to far-red. Fluorescent proteins: Nuclear behavior can be visualized dynamically in live cells using CellLight® nuclear and histone constructs. CellLight® reagents express fusion proteins with red or green fluorescence for imaging in live cells or after formalin fixation. Antibody conjugates: With Molecular Probes® antibody conjugates you can label nuclear components in fixed cells or frozen tissue sections even after paraffin embedding. (Unconjugated antibodies require the use of secondary detection labels.) SYTOX® dyes: SYTOX® dyes stain nucleic acids in fixed cells with high affinity and a 500x signal enhancement on binding. They can easily be multiplexed and serve as dead cell stains. For more information go to lifetechnologies.com/nucleus For research use only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO0 013 Fluorescence for studying morphology and outgrowth Cell tracing tools Cell morphology and connectivity are key components in understanding neuronal function. Connectivity and function are routinely studied in cultured cells, fixed tissue sections, and live tissues that are subsequently fixed for analysis. The fluorescence technologies used to trace neural networks can be divided into those for single cells or cell populations. While the different classes of probes listed here all label tissue sections and cultured cells, there are unique performance features for addressing each specific application and answering each biological question. •Bright fluorescence for visualizing fine neuronal processes •Choice of technologies for optimized performance in any application •Choice of colors for multiplexing with other fluorescent reporters Hydrazides and biocytins: Bright polar tracers with multiple colors are loaded into cells by microinjection. They provide fixable and photostable cellular contrast and also track gap junctions. Conjugated dextrans: Available in a range of colors and sizes and loaded by microinjection. Smaller dextrans will track gap junctions, while larger dextrans are slower and more persistent for axonal tracing. Lipophilic tracers: Formulated as crystals or pastes for applying directly to tissues, lipophilic tracers will stain fresh or fixed tissue sections as well as live cells, and track neuronal outgrowths in both directions. Toxins and lectins: These fixable conjugates all have unique properties and a range of fluorescent reporters. Wheat germ agglutinin is a retrograde tracer, while cholera toxin is anterograde. IB4 differentiates between different classes of neurons. For more information go to lifetechnologies.com/neuronaltracers For research use only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO07205 0113 Studying migration, chemotaxis, and invasion Cell tracking tools Cell migration studies are key to understanding processes such as wound healing, immune response, and tumor metastasis. Ideal tracking tools should be bright enough for easy visualization, should resist cell-to-cell transfer, and for longterm tracking, should remain in the cell for the required number of divisions. Molecular Probes® products offer solutions based on transgenic protein expression, organic dyes, or Qdot® probes. Each solution is available for a range of applications with several wavelengths for multiplexed detection. •Bright fluorescence for easy detection and quantitation •Choice of technologies for optimized performance in any application •Choice of colors for multiplexing with other fluorescent reporters CellLight® solutions Transiently transduced cells express fluorescent fusion proteins at different locations within the cell. Labeled cells are easy to track for at least five days or up to two weeks in slow-growing populations. Learn more at lifetechnologies.com/celllight CellTracker™ solutions Organic dyes will label cell populations easily without the need for transduction. CellTracker™ probes provide bright, uniform labeling of cells in a range of colors lasting for 3 to 6 generations. Learn more at lifetechnologies.com/celltracker Qtracker® solutions Qtracker® probes are extremely bright and resistant to photobleaching. They provide punctate labeling of cells in a wide range of wavelengths that lasts for 6 to 10 generations of tracking. Learn more at lifetechnologies.com/qtracker For more information go to lifetechnologies.com For research use only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO07205 0113 Tracking the endocytic pathway with fluorescence Phagocytosis and endocytosis are the processes by which cells internalize particulate matter such as microorganisms, and materials such as proteins and ligands. They are important in immune responses and the clearance of apoptotic cells in metabolism and cell signaling. When cells internalize proteins or particles, the endosome is acidic relative to the extracellular environment, so pH indicators like pHrodo™ dye are useful for monitoring stages in the pathway from early endosome to lysosome formation. •Monitor phagocytosis with red or green fluorescence •Sensitive and robust assay with no washing or quenching required •Flexible workflow for imaging, flow cytometry, or microplate assays RAW cells were incubated with zymosan BioParticles® particles (containing pHrodo™ Green dye) and were imaged in Live Cell Imaging Solution (Cat. No. A14291DJ). Green fluorescence indicates that the particles have become acidified. Schematic of pHrodo™ dye–based detection of phagocytosis and endocytosis. Particles (microorganisms or proteins) labeled with pHrodo™ dye are added to cells. Some remain in solution or become nonspecifically attached to cells—they do not fluoresce because of the neutral pH of the extracellular environment. Some are taken up by cells by phagocytosis or endocytosis and become encapsulated in vesicles. As vesicles are processed, pH decreases and the pHrodo™ dye–labeled particles fluoresce. Phagocytosis Endocytosis and pinocytosis Selection guide Application Phagocytosis Red Green Dextran (10K) Zymosan P35364 P35365 E.coli P35361 P35366 S. aureus A10010 Endocytosis Red Green P10361 P35368 P35367 Reactive pHrodo dyes allow you to conjugate your own particles or proteins Coupling chemistry Red Avidin P35362 Maleimide P35371 SE P35363 STP ester For more information, go to lifetechnologies.com/phrodo For research use only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO07205 0113 Green P35370 P35369 Monitoring oxidative stress and reactive oxygen species CellROX® reagents Oxidative damage is a fact of life associated with aging, disease, and cellular damage. To measure oxidative stress in live cells, CellROX® reagents generate a fluorescent green, orange, or deep red signal. You can use flow cytometry, fluorescence imaging, or microplate fluorometry to monitor the signal and easily multiplex with immunostaining or with other fluorescent reporters. •Monitor reactive oxygen species with deep red, orange, or green fluorescence •Flexible workflow for imaging, flow cytometry, or microplate assays •Choice of colors for multiplexing with other fluorescent reporters Imaging oxidative stress with CellROX® Green Reagent. Human osteosarcoma (U2-OS) cells expressing CellLight® Actin-RFP were treated with 100 µM menadione. Cells were stained with CellROX® Green Reagent (Cat. No. C10444) and NucBlue™ Live Cell Stain (Cat. No. R37605), washed, and imaged with Live Cell Imaging Solution (Cat. No. A14291DJ). CellROX® reagent selection guide Ex/Em max Deep red Orange Green 640/665 nm 545/565 nm 485/520 nm Live-cell compatible Yes Yes Yes Can be added to complete media Yes Yes Yes Formaldehyde fixable Yes No Yes Multiplex ready No No Yes Signal to noise +++++ +++++ +++++ Photostability +++ ++++ ++ GFP compatible No Yes Yes RFP compatible Yes No Yes Cat. No. C10422 C10443 C10444 For more information go to lifetechnologies.com/cellrox For research use only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO07205 0113 Autophagy—cells under stress Fluorescence-based tools for LC3B detection The LC3B protein plays a critical role in autophagy. Normally, LC3B resides in the cytosol, but following cleavage and lipidation with phosphatidylethanolamine, the LC3B protein associates with the phagophore and can be used as a general marker for autophagic membranes. A series of Molecular Probes® fluorescence-based tools are available to help visualize and detect this critical protein. •Premo Autophagy Sensor—detect LC3B temporally and spacially in live cells ™ •LC3B Antibody Kit for Autophagy—complete kit to visualize LC3B localization in fixed samples •Premo™ Autophagy TR-FRET Assay—obtain quantitative, plate reader measurement of autophagy Mitochondrion Protein Amino acids, fatty acids Peroxisome Phagophore Nucleus Autolysosome Autophagosome LC3B recruitment LC3B disassociation Lysosome Schematic depiction of the multistep autophagy pathway in a eukaryotic cell. The first step involves the formation and elongation of isolation membranes, or phagophores. In the second step, which involves the LC3B protein, the cytoplasmic cargo is sequestered, and the double-membrane autophagosome is formed. Fusion of a lysosome with the autophagosome to generate the autolysosome is the penultimate step. In the fourth and final phase, the cargo is degraded. Premo™ Autophagy Sensor The Premo™ Autophagy Sensor combines the selectivity of an LC3B-fluorescent protein (LC3B-FP) chimera, with the transduction efficiency of BacMam technology, enabling unambiguous visualization of this protein in live cells. Available in either GFP or RFP configurations, the reagent also tolerates fixation with formaldehyde and thus is compatible with fixed-cell analysis, which is required for multiplex experiments using antibodies, and is preferred for HCS analysis workflows. The LC3B Antibody Kit Each LC3 Antibody Kit for autophagy includes a rabbit polyclonal antibody against LC3B that has been validated for use in fluorescence microscopy and high-content imaging and analysis. The kit also includes a control compound for inducing autophagosomes—following treatment with this compound, lysosomal pH increases and the normal autophagic flux is disrupted, resulting in autophagosome accumulation. Premo™ Autophagy TR-FRET Assay The Premo™ Autophagy TR-FRET Assay measures changes in the autophagy activity of cells expressing GFP-tagged LC3B using a Terbium (Tb)-based TRFRET plate reader immunoassay approach, providing you with a quantitative, objective measurement of autophagy. The expression kit provides BacMam reagent for easily expressing GFP-LC3B in many different cell types along with a Tb-labeled LC3B-II specific detection antibody for performing the assay. For more information go to lifetechnologies.com/autophagy For research use only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO07205 0113 Apoptosis—caspases bringing cell death to light CellEvent™ Caspase-3/7 detection reagent Cysteine aspartic acid proteases, or caspases, are enzymes that play a key role in initiating and executing apoptosis. Activation of caspases is one of the more definitive markers of programmed cell death. The CellEvent™ Caspase-3/7 Green detection reagent is a new fluorogenic substrate for activated caspases 3 and 7 that is compatible with microscopy, flow cytometry, or high-content imaging, and is well suited for multiplex imaging in live or fixed cells. •Optimized caspase-3⁄7 substrate for apoptosis analysis by microscopy, flow cytometry, or highthroughput screening untreated 2-hr staurosporine 4-hr staurosporine Traditional fluorescence imaging of oxidative stress and apoptosis. HeLa cells were treated with 0.5 µM staurosporine for 0, 2, or 4 hr in the presence of 7.5 µM CellEvent™ Caspase-3/7 Green detection reagent. Cells were then stained with 5 µM CellROX ™ Deep Red reagent and Hoechst 33342 nucleic acid stain for 30 min at 37°C, washed with warm DPBS, and imaged immediately. Increased oxidative stress was observed at 2 hr after staurosporine treatment (magenta), whereas caspase-3/7 activation was not observed until 4 hr after treatment (green). •Simple, no-wash protocol helps preserve delicate apoptotic cells green nuclei, while cells without activated caspase 3/7 show minimal fluorescence. •Compatible with both live cell fluorescence imaging and formaldehyde-based fixation methods This robust assay is highly specific for caspase 3/7 activation and, as expected, nearly complete inhibition of the CellEvent™ Caspase-3/7 Green detection reagent signal was observed after pretreatment of cells with Caspase 3/7 Inhibitor 1. The fluorescent signal from the CellEvent™ Caspase-3/7 reagent survives fixation and permeabilization, providing the flexibility to perform end-point assays and probe for other proteins of interest using immunocytochemistry. The CellEvent™ detection reagent requires no wash steps—simply add it to cells in complete culture medium and incubate for 30 minutes. Samples are ready for imaging using the standard FITC filter set. Apoptotic cells with activated caspase 3/7 show bright Product CellEvent Caspase-3/7 Detection Reagent ™ Platform Flow cytometry laser Imaging filter set Ex/Em (nm) Cat No. FC,I,HCS,HTS Blue (488 nm) FITC 502/530 C10423 To see CellEvent™ Caspase-3/7 reagent in action, go to lifetechnologies.com/celleventvideo For research use only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO07205 0113 Apoptosis—DNA fragmentation Click-iT® TUNEL Assays—the best and the brightest Click-it® TUNEL is a breakthrough technology using click chemistry for detection of DNA fragmentation. For TUNEL assays to yield meaningful results, it's necessary that the modified nucleotide is an acceptable substrate for TdT, and that the detection method is sensitive and avoids detrimental loss of cells from the sample. Click-it® TUNEL Assays: •Detect late-stage apoptosis before cells "pop" •Make it easy to multiplex with additional apoptosis markers •Save you time with click chemistry How the TUNEL assay works with click chemistry Click-iT® TUNEL Imaging Assays The Click-iT® TUNEL Imaging Assays employ dUTP modified with an alkyne, a small, bio-orthogonal functional group. The incorporated nucleotide is detected through click chemistry—a copper-catalyzed reaction between an azide and an alkyne. Click chemistry Click chemistry is an alternative way to label and detect when methods such as direct labeling or using antibodies are too destructive to the sample. The small size of the Alexa Fluor® azide (MW ~1,000) compared to that of an antibody (MW ~150,000) enables effortless penetration of complex samples with only mild fixation and permeabilization needed. DNA strand breaks typical of late-stage apoptosis visualized using the Click-iT® TUNEL Imaging Assay (red) on HeLa cells treated with staurosporine. Activated caspase-3 was detected with a rabbit polyclonal primary antibody for cleaved caspase-3 and labeled with Alexa Fluor® 488 goat anti–rabbit IgG (green). Tubulin was detected with a mouse monoclonal anti-tubulin antibody and labeled with Alexa Fluor® 555 goat anti–mouse IgG (orange). Nuclei were stained with Hoechst 33342 (blue). The cells on the right not only have a high level of caspase-3 activity and DNA strand breaks, but also show a loss of structural integrity consistent with cells undergoing apoptosis. Click-iT® TUNEL click chemistry Alexa Fluor® detection Available in three different bright and photostable Alexa Fluor® dyes To unequivocally establish programmed cell death in any given cell or tissue model, two or more biomarkers of apoptosis must be observed. The click chemistry Click-iT® TUNEL Imaging Assays are available in three different bright and photostable Alexa Fluor® dyes, ranging from green to far-red fluorescence, providing flexibility when working with other apoptosis detection reagents that are typically available only in green- or red-fluorescent colors. Detects DNA fragmentation in adherent cells Each Click-iT® TUNEL kit contains all of the components necessary to accurately and reliably detect DNA fragmentation with simple click chemistry reactions in adherent cells grown on coverslips or in 96-well microplates. Product Platform Common imaging filter sets Ex/Em (nM) Cat No. Click-iT® TUNEL Alexa Fluor® 488 Imaging Assay I, HCS FITC 495/519 C10245 Click-iT TUNEL Alexa Fluor 594 Imaging Assay I, HCS Texas Red 590/615 C10246 Click-iT® TUNEL Alexa Fluor® 647 Imaging Assay I, HCS Cy® 5 650/670 C10247 ® ® For more information go to lifetechnologies.com/tunel For research use only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO07205 0113 Simple, efficient, effective cell viability determination Cell viability—LIVE/DEAD® viability assays for mammalian cells Molecular Probes® LIVE/DEAD® assays provide complete solutions for easy, sensitive determination of cell viability, cell vitality, and compound cytotoxicity. These fluorescence-based assays can be used to examine a variety of mammalian cell types. Specific LIVE/DEAD® assays can be used for flow cytometry, microscopy, or microplate formats. Principle of assays LIVE/DEAD® Viability/Cytotoxicity Kit (Cat. No. L3224)— quick and easy two-color assay to determine viability of cells in a population based on plasma membrane integrity and esterase activity. • Live stain: calcein AM (green) Viability of kangaroo rat (PtK2) cells using the LIVE/DEAD® Viability/ Cytotoxicity Kit. Live and dead PtK2 cells were stained with ethidium homodimer-1 and the esterase substrate calcein AM, both provided in the LIVE/DEAD® Viability/Cytotoxicity Kit . Live cells fluoresce a bright green, whereas dead cells with compromised membranes fluoresce red-orange. • Dead stain: ethidium homodimer-1 (red) LIVE/DEAD® Cell-Mediated Cytotoxicity Kit (Cat. No. L7010)— measures cytotoxicity mediated by natural killer cells, lymphokine-activated killer cells, and T cells. • Live stain: DiOC18(3) (green) • Dead stain: propidium iodide (red) LIVE/DEAD® Sperm Viability Kit (Cat. No. L7011)— analyzes the viability and fertilizing potential of sperm by fluorescence microscopy or flow cytometry. • Live stain: SYBR® 14 dye (green) • Dead stain: propidium iodide (red) LIVE/DEAD® Cell Vitality Assay Kit (Cat. No. L34951)—simple, two-color fluorescence assay that distinguishes metabolically active cells from injured cells and dead cells. Distinguishing live from dead sperm with the LIVE/DEAD® Sperm Viability Kit . Live sperm with intact membranes were labeled with SYBR® 14 dye, a proprietary cell-permeant nucleic acid stain, and fluoresce green. Dead sperm, which were killed by unprotected freezethawing, were labeled with propidium iodide and fluoresce red-orange. Both dyes are components of the kit. The image was contributed by Duane L. Garner, School of Veterinary Medicine, University of Nevada, Reno, and Lawrence A. Johnson, USDA Agricultural Research Service. • Live stain: C12-resazurin (red) • Dead stain: SYTOX® Green (green) For more information go to lifetechnologies.com/livedeadassays For research use only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO07205 0113 Live-cell imaging of cell cycle and division Solutions for microscope imaging and HCS Measuring a cell’s ability to proliferate and exploring the mechanisms of cell growth are fundamental methods used in developmental and stem cell biology, cancer research, and drug discovery and toxicology. Several Molecular Probes® assays are now available to efficiently and accurately measure the progression of a cell through the cell cycle, whether you are exploring the mitotic pathway or evaluating a novel compound. Cell cycle measurement in live cells Premo™ FUCCI Cell Cycle Sensor is a fluorescent two-color sensor of cell cycle progression and division in live cells. It allows accurate and sensitive cell cycle analysis of individual cells or a population of cells by fluorescence microscopy, flow cytometry, or high-content imaging. The fluorescence ubiquitination cell cycle indicator (FUCCI) is a genetically encoded, two-color (red and green) indicator that lets you follow cell division within a cell population. We have incorporated the FUCCI genetic constructs into the powerful BacMam gene delivery system, creating a simple and efficient method for labeling cells and following their division. • Accurate—monitor changes in cell proliferation with direct visual readout of cell cycle phase for every cell in the population • Highly efficient—>90% transduction of a wide range of mammalian cell lines, including primary cells and stem cells Visualization of cell cycle phases with the Premo™ FUCCI Cell Cycle Sensor over a 16-hour period. Initially, the cell in the center of the image transitioned from green (G2/M) to red (G1) to yellow (S). Approximately 6 hr after imaging was initiated, the cell at the top of the visual field migrated down before undergoing mitosis (green) and progressing into G1 (red). Imaging cell cycle progression in live cells with Premo™ FUCCI Cell Cycle Sensor. Schematic of cell cycle progression with nuclear fluorescence changes. • Fast and convenient—simply add Premo™ FUCCI Cell Cycle Sensor to your cells in complete medium, incubate overnight Ordering information Product Quantity Premo™ FUCCI Cell Cycle Sensor (BacMam 2.0) 1 kit For more information, go to lifetechnologies.com/fucci For research use only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO07205 0113 Cat. No. P36238 Cell proliferation and DNA replication Multicolor imaging techniques for accurate cell proliferation assays Cell proliferation assays are important tests to determine whether a cell population is actively dividing or has undergone division during an assay period. Using fluorescent labels to detect new DNA synthesis can provide rich data sets on imaging and microplate platforms. With a choice of fluorescent labels, assays can be multiplexed to explore other cell health parameters and answer complex biological questions. •Superior performance—highly sensitive assays to detect cell division •Flexibility—multiple assays and colors available for multiparametric studies •Efficiency—quick, robust, easy-to-use labeling techniques for multiple cell types Stains for new DNA synthesis The Click-iT® EdU Assay kits provide simple, robust methods for analyzing DNA replication in proliferating cells in as little as 90 minutes. Kits are available for microscopic imaging or high-content screening (HCS) analysis, with a choice of fluorescence wavelengths for multiplexing or with Amplex® UltraRed signal amplification to provide a fluorescence intensity readout on microplates. Multicolor imaging with Click-iT® EdU. Muntjac cells pulsed with EdU, fixed and permeabilized. Labeled DNA was detected using the Click-iT® EdU Alexa Fluor® 647 Imaging Kit (Cat. No. C10340). Tubulin (in blue) is labeled with a mouse primary followed by Alexa Fluor® 350 goat anti–mouse IgG antibody. Golgi (in green) is labeled with Alexa Fluor® 488 conjugate of lectin HPA, and peroxisomes (in orange) are labeled with a rabbit primary followed by Alexa Fluor® 555 donkey anti–rabbit IgG antibody. Ordering information Product Size Cat. No. Click-iT® EdU Microplate Assay 1 kit/400 assays C10214 Click-iT® EdU Alexa Fluor® 488 HCS Assay 2 plates C10350 Click-iT® EdU Alexa Fluor® 555 HCS Assay 2 plates C10352 Click-iT® EdU Alexa Fluor® 594 HCS Assay 2 plates C10354 Click-iT® EdU Alexa Fluor® 647 HCS Assay 2 plates C10356 Click-iT® EdU Imaging Kit with Alexa Fluor® 488, 594, and 647 1 kit C10086 Click-iT® EdU Alexa Fluor® 488 Imaging Kit 1 kit/50 coverslips C10337 Click-iT® EdU Alexa Fluor® 555 Imaging Kit 1 kit/50 coverslips C10338 Click-iT® EdU Alexa Fluor® 647 Imaging Kit 1 kit/50 coverslips C10340 For more information, go to lifetechnologies.com/clickit For research use only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO07205 0113 ReadyProbes™ ready-to-use imaging reagents No Calculations. No Dilutions. No Resuspensions. Fluorescence imaging has never been easier with ReadyProbes™ reagents. • Dropper bottle formats See the following example of a simple ReadyProbes™ protocol. Protocol for NucBlue® Live Cell Stain • Stable at room temperature • Simplified protocols • No dilutions, resuspensions, calculations, or aliquoting necessary 1. Add 2 drops per mL of medium 2. Incubate 5–20 min at 20–25ºC 3. Image cells ActinGreen™ 488 ReadyProbes™ reagent. BPAE cells were fixed, permeabilized, and blocked using the Image-iT® Fixation/ Permeabilization Kit. Tubulin was labeled using an anti-tubulin antibody followed by detection with an Alexa Fluor ® 594 secondary. Actin was stained using ActinGreen™ 488 ReadyProbes™ reagent and nuclei were stained with NucBlue® Fixed Cell Stain. Available Reagents Product Nuclear stains NucBlue® Live ReadyProbes™ Reagent NucBlue® Fixed Cell ReadyProbes™ Reagent NucGreen™ Dead 488 ReadyProbes™ Reagent Propidium iodide ReadyProbes™ Reagent NucRed™ Live 647 ReadyProbes™ Reagent NucRed™ Dead 647 ReadyProbes™ Reagent Actin stains ActinGreen™ 488 ReadyProbes™ Reagent ActinRed™ 555 ReadyProbes™ Reagent Apoptosis reagent CellEvent® Caspase 3/7 Green ReadyProbes™ Reagent Imaging accessories BackDrop® Background Suppressor ReadyProbes™ Reagent Live Cell Imaging Solution* Image-iT® Fixation/Permeabilization Kit* Image-iT® FX Signal Enhancer ReadyProbes™ Reagent *Not available in dropper bottle format Filter Ex/Em (nm) Cat. No. DAPI DAPI FITC RFP Cy®5 Cy®5 358/461 352/461 504/523 535/617 638/686 642/661 R37605 R37606 R37109 R37108 R37106 R37113 FITC RFP 495/518 540/565 R37110 R37112 FITC 495/518 R37111 NA NA NA NA NA NA NA NA R37603 A14291DJ R37602 R37107 Find out more at lifetechnologies.com/readyprobes For Research Use Only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO07205 0313 Antifade reagents Minimize photobleaching across the visible spectrum Loss of fluorescent signal through irreversible photobleaching processes can lead to a significant reduction in sensitivity, particularly when target molecules are of low abundance or when excitation light is of high intensity or long duration. To minimize photobleaching of experimental samples we have developed a series of antifade reagents that increase the photostability of many popular fluorophores and preserve signals for higher sensitivity and extended analysis. •Inhibit photobleaching across the spectrum •Mounted samples stable for weeks to months Both SlowFade® and ProLong® reagents are available preformulated with DAPI as a nuclear counterstain, eliminating the need for a separate counterstaining step. •Available with or without DAPI counterstain Convenience •Hard-setting or immediate-use formulations •Ready-to-use benchtop formulation •Optimized refractive index Choosing the right antifade Life Technologies antifade reagents are optimized for a variety of sample requirements such as longterm storage or immediate imaging. SlowFade® Antifade Reagent provides immediate imaging and short-term (3–4 weeks) storage, while ProLong® Antifade Reagent requires a curing period but provides long-term stability. Life Technologies antifade reagents are now formulated for room temperature storage and provided in dropper bottles for immediate benchtop use. Green chemistry As well as providing improved convenience for routine imaging, these reagents provide energysaving room temperature storage. Environmental and health risks have also been significantly reduced by the elimination of sodium azide. ProLong® Gold Antifade Reagent SlowFade® Gold Antifade Reagent Long-term storage Yes Yes Yes Yes No No No No Immediate viewing No No No No Yes Yes Yes Yes Counterstain No No DAPI DAPI No No DAPI DAPI Quantity 5 x 2 mL 10 mL 5 x 2 mL 10 mL 5 x 2 mL 10 mL 5 x 2 mL 10 mL Cat. No. P36934 P36930 P36935 P36931 S36937 S36936 S36939 S36938 View all products online at lifetechnologies.com/antifades For Research Use Only. Not for use in diagnostic procedures. ©2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO07205 0613