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Live-Cell ImagingLearn the simple steps to stain your live cells for dynamic imaging experiments. |
What is fluorescence live-cell imaging?
Fluorescence imaging of live cells is used to observe dynamic cellular processes and track cellular biomolecules and structures over time. Live-cell fluorescence imaging allows researchers to examine cells in their native state so that cellular function is preserved, enabling the study of cellular processes including cytoskeletal rearrangement, apoptosis, cell migration, endocytosis, phagocytosis, and organelle dynamics to obtain more relevant findings in drug discovery, cancer, developmental biology, environmental science, and other medical research fields.
Use this guide to build live-cell imaging experiments, pick reagents, and generate publication quality data.
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How to choose reagents for live-cell imaging
Live-cell imaging reagents include both targeted fluorescent proteins and small membrane permeant fluorescent dyes. To enable live-cell imaging experiments, many reagents are designed for time-lapse over several hours or days, while others are optimal for end-point assays in which cells are imaged and analyzed immediately after staining. Staining concentration, incubation time, and the appropriate time window and imaging interval/frequency should be determined empirically to minimize cytotoxicity and preserve cellular function.
The reagents found within the live-cell imaging guide below are compatible with automated high-content and incubator-based fluorescence imaging systems such as: Thermo Fisher Scientific’s EVOS M7000 and M5000 Cell Imaging Systems with EVOS Onstage Incubator, CellInsight HCS Platforms, PerkinElmer’s MuviCyte™ live-cell imaging system*, Leica’s Thunder and Mica*, and Sartorius’ Incucyte® Live-Cell Analysis System™*.
How to maintain environmental control during live-cell imaging
Fluorescence imaging platforms combined with incubators provides a controlled and stable environment that allows long-term imaging of cells in their natural state. The EVOS Onstage Incubator for the EVOS M7000 and M5000 Cell Imaging Systems and the Invitrogen HCA Onstage Incubator for automated high-content screening and analysis on the CellInsight HCS/HCA platforms enable time-lapse and kinetic imaging and analysis of live cells within an environmental chamber. When performing long-term live-cell imaging within an incubated environment, reagents should be used in traditional cell culture medium or FluoroBrite DMEM (Cat. No. A1896701) buffered with bicarbonate to maintain physiological pH in a 5% CO2 environment.
Example: apoptosis in live-cell imaging experiments
Cellular apoptosis is a tightly controlled programmed cell death process used to remove excess, damaged, and unneeded cells or tissues. Apoptosis can be detected through caspase activation, DNA fragmentation, disruption in active mitochondria, and plasma membrane changes. The CellEvent Caspase 3/7 reagents are fluorogenic caspase substrates that detect caspase activation to assay for apoptosis in live cells. These reagents do not require wash steps and can be used for real-time and time-lapse live-cell imaging of apoptosis for 48 hours or longer.
Figure 1. Apoptosis live-cell endpoint assay workflow using the CellEvent Caspase-3/7 Green Detection Reagent (Cat. No. C10432). Seed cells and follow with treatment. Add diluted CellEvent Caspase-3/7 Green Detection Reagent to cells. CellEvent Caspase 3/7 dyes may be imaged directly on cells in complete media without wash. Measure fluorescence starting as early as 30 minutes to as long as 72 hours.
Figure 2. Example of Live Cell Imaging of apoptotic cells stained with CellEvent Caspase 3/7 Green. HuVEC cells treated with a cocktail stained with CellEvent Caspase 3/7 Green Assay (Cat. No. C10432) and imaged with Incucyte® ZOOM Live-Cell Analysis System at 0, 24, 48, and 72 hours after treatment. (Image from: Sambi M, Samuel V, Qorri B, Haq S, Burov SV, Markvicheva E, Harless W, Szewczuk MR. A Triple Combination of Metformin, Acetylsalicylic Acid, and Oseltamivir Phosphate Impacts Tumour Spheroid Viability and Upends Chemoresistance in Triple-Negative Breast Cancer. Drug Des Devel Ther. 2020;14:1995-2019 PMID: 32546966)
Example: mitochondrial activity in live-cell imaging experiments
Live-cell imaging reagents include both cell structure reagents used to identify cellular components and cell function reagents to analyze cellular functions and processes. For instance, MitoTracker dyes accumulate in active mitochondria in live cells and covalently attach to mitochondria to allow evaluation of mitochondrial localization and abundance. In contrast, TMRM and TMRE are dynamic mitochondria membrane potential indicator dyes that fluctuate in and out of the mitochondria based on membrane potential.
Figure 3. Monitoring active mitochondria in live cells. Fibroblast lines from patients were cultured and stained with MitoTracker Red (Cat. No. M22426), Image-iT DEAD Green dyes (Cat. No. I10291), Hoechst (Cat. No. R37165) and imaged on an Incucyte® ZOOM Live-Cell Analysis System. (Image from Smith GA, Jansson J, Rocha EM, Osborn T, Hllett PJ, Isacson O. Fibroblast Biomarkers of Sporadic Parkinson's Disease and LRRK2 Kinase Inhibition. Mol Neurobiol. 2016 Oct;53(8):5161-77. PMCID: PMC5012155. )
Example: viability in live-cell imaging experiments
Cell impermeant nucleic acid stains can be used as dead cell indicators in live-cell imaging experiments. Nucleic acid stains such as YOYO-1 and YOYO-3 are nontoxic dyes that have bright signals and large fluorescence enhancement upon binding to DNA. These dyes are impermeant to viable cells and selectively stain dead cells with compromised membranes to enable live-cell imaging over time.
Figure 4. Monitoring cell viability with YOYO-3. Mouse embryonic fibroblasts (MEF) were treated with cycloheximide (CHX) and stained with YOYO-3, then scanned at 2-hour intervals for up to 24 hours on the Incucyte® ZOOM Live-Cell Analysis System to monitor cell viability based on dead cell staining. Annexin V and CellEvent Caspase 3/7 Green were also used to detect apoptotic cells. (Image from Gelles JD, Chipuk JE. Robust high-throughput kinetic analysis of apoptosis with real-time high-content live-cell imaging. Cell Death Dis. 2016 Dec 1;7(12):e2493. doi: 10.1038/cddis.2016.332. Erratum in: Cell Death Dis. 2017 May 4;8(5):e2758. PMCID: PMC5261025.)
Tips to detect changes in live-cell experiments
Step 1: Plan
Design your experiment with careful consideration of the tools and resources needed for each step.
Advantages
- Observe dynamic cellular processes as they happen
- Study and image several processes and functions simultaneously using multiplexed assays
- Study cellular structures in their native environment, resulting in more realistic results closer to in vivo scenarios
- Track cellular biomolecules and structures over time
- Observe interactions between cells
- Cellular enzymes and other cytosolic biomolecules remain in the cell
Considerations
- Must have a specific way to label your target with minimal toxicity – whether it is a molecule, a cellular function, or a cellular state
- Living cells are generally not permeable to large detection molecules such as antibodies
- Moving objects can be more difficult to keep in focus
- Certain techniques can be harmful to living cells
- Cells must be kept in their natural physiological state
Step 2: Culture
Maintain or grow your cells in optimum conditions.
Keeping cells alive and healthy during various experimental manipulations, detection, and imaging is no small task. The choice of medium is particularly important for time-lapse imaging and experiments where cells are exposed to ambient conditions for longer periods. For reliable results with live cells, it is essential that the cells be healthy and kept in an environment as close as possible to physiological temperature, pH, oxygen level, and other conditions.
Product highlights
These media and wash buffers are created specifically for live-cell imaging and detection. Employing them in your experiments can help you improve image clarity, reduce background fluorescence, and optimize cell viability.
- Thermo Scientific Nunc cell culture vessels with Nunclon Delta surface treatment have been validated with Gibco media to confirm consistent cell growth across multiple cell lines. It’s a proven combination for happy cells and happy scientists.
- Gibco extracellular matrices, scaffolds, and proteins provide in vivo–like morphology and physiologically relevant environments for more realistic cell biology and better intercellular interactions.
- Invitrogen Countess 3 FL Automated Cell Counter is an affordable and automated tool for checking the health of your cells quickly and objectively. It has reusable or disposable slide options.
TipsYou can improve image clarity, reduce background fluorescence, and optimize cell viability by using media and wash buffers created specifically for live-cell imaging and detection. See product selection guide |
Step 3: Label
Target cell structures, cell functions, and proteins of interest with selective dyes and stains.
The appropriate fluorophore (targeted fluorescent protein or small membrane-permeant reagent) should be used to monitor your target cellular structure or process. Additional fluorophores can be used to monitor multiple cellular structures and processes, but the excitation and emission spectra should be checked using the Fluorescence SpectraViewer or Stain-iT Cell Staining Simulator to ensure minimal spectra overlap. It is critical to avoid using too much fluorescent label because excessive fluorescent labeling can result in:
- Nonspecific staining with increased background signals
- Physiological artifacts and structural perturbations
- Cytotoxicity
- Spectral overlap
Note that:
- Live-cell structure reagents—help identify cellular components
- Live-cell function reagents—help identify cellular functions and processes
Product highlights
- Invitrogen CellLight reagents have proven to be the easiest to use for labeling specific structures in live cells. Targeted fluorescent proteins are introduced using the Invitrogen BacMam transduction system; no molecular biology techniques are required. Simply add the reagent to your cells, incubate overnight, and you’re ready to image in the morning.
- Invitrogen CellTracker reagents are a diverse reagent class used for labeling mammalian cells to view changes in morphology or location. These nontoxic fluorescent dyes are designed to freely pass through cell membranes into cells, where they are transformed into cell-impermeant reaction products. Incubating cells with a CellTracker reagent for 30 minutes will provide at least 72 hours of fluorescent signal (typically three to six generations).
- Invitrogen pHrodo indicators are fluorogenic dyes that dramatically increase in fluorescence as the pH of their surroundings becomes more acidic. When conjugated to dextrans, proteins, or other particles, pHrodo dyes can be used as highly specific sensors of endocytic and phagocytic internalization and lysosomal sequestration in live cells, offering a superior alternative to conjugates of other fluorescent dyes such as fluorescein and tetramethylrhodamine.
Tips
|
Step 4: Optimize
Minimize background and maintain photostability of fluorescence signals.
Signal-to-background ratio can be optimized by using reagents that reduce extracellular fluorescence and increase fluorophore photostability. It is important to image in media that have been specifically designed for maintaining cell health while reducing or eliminating background fluorescence in live-cell imaging experiments (see Table 1). The addition of a background suppressor compatible with live cells can also help reduce extracellular background fluorescence and eliminate the need for a wash step. ProLong Live Antifade Reagent can be applied to samples to reduce photobleaching of fluorophores, preventing signal loss with multiple or long exposures.
Table 1. Imaging media comparison.
| Reagent | Cell washing | Short-term imaging | Imaging up to 4 hours | Long-term imaging |
|---|---|---|---|---|
| Gibco PBS, pH 7.4 |  | | ||
| Gibco FluoroBrite DMEM | | | | |
| Invitrogen Live Cell Imaging Solution | | | |
Product highlights
- Invitrogen BackDrop Background Suppressor is used when observing high background signal or weak fluorescence in the blue, green, or red channels.
- Gibco PBS, pH 7.4 is ideal for cell washing and short-term imaging where prolonged incubation is not required.
- Invitrogen Live Cell Imaging Solution is an optically clear solution used for imaging, dye loading, and wash steps. It helps keep cells healthy for up to 4 hours.
Live HeLa cells labeled with Tubulin Tracker Green dye and Tubulin Tracker Deep Red dye. Both labels show high off-cell background when the probe is left in the staining solution (left). Addition of BackDrop Background Suppressor greatly reduces extracellular background while leaving intracellular labeling unaffected (right), thus enabling a no-wash protocol for high-contrast imaging of tubulin in live cells.
The overall signal protection offered by ProLong Live reagentcompared to untreated samples is calculated based on the scan number where treated and untreated samples reach the EC50 value. The addition of ProLong Live reagent permitted 100% more captures with Invitrogen CellLight Mitochondria-RFP reagent.
After 120 exposures using a standard time-lapse imaging protocol, samples treated with ProLong Live reagent are >20% brighter than untreated cells, enabling more data collection time.
Tips
|
Step 5: Image
Live-cell imaging of dynamic processes requires active observation over time
Illumination and detection
To minimize phototoxicity, choose imaging systems that give you the greatest control of light sources. Try to minimize light intensity, exposure time, wavelength range, and amount of excitation energy for illuminating your cells while still generating a good signal with low background. Use the illumination that gives you the highest signal with the lowest level of fluorophore excitation. In some cases (particularly when you wish to image over a long period of time), it is advisable to sacrifice resolution by using shorter exposure times or lower magnification in exchange for healthier cells.
Live-cell imaging over longer periods of time can be challenging because the target may move out of focus during the course of the experiment. Many microscopes have autofocusing features that can help keep your target in focus longer and reduce focal drift. Additionally, maintaining cells at a constant temperature and keeping the volume of solution in the vessel constant will help with focal drift.
Environmental control
Many cells cannot tolerate deviations from their optimal temperature, osmolarity, pH, and humidity. Requirements vary depending on what experimental question you are asking. For example, experiments investigating cell growth and division may have a different set of requirements than experiments involving receptor activation and calcium accumulation. Some robust immortalized cell lines will tolerate being imaged or monitored for short periods of time without any environmental control. Conversely, for long-term imaging and detection studies, good results with both immortalized cells and primary cells typically require tightly controlled environmental parameters.
Click image to enlarge
A scratch wound in a culture of HDFn cells loaded with Invitrogen CellTracker Deep Red Dye. (A) The illuminated area was subjected to repeated illumination for 10 hours. Cells in this area show signs of phototoxicity (a loss of viability as cells were not able to grow into the wound). (B) Cells in the non-illuminated area show viable cell growth into the wound.
The top cell shows catastrophic blebbing of the cell membrane caused by excessive light exposure. Blebbing is a term used to describe membrane perturbation caused by toxicity. By contrast, the bottom cell remains relatively healthy and is not displaying aberrant morphology.
Product highlights
To avoid the pitfall of proceeding to the next step in your experiment with unhealthy cells, a quick check for cell health can be done on the Countess 3 FL Automated Cell Counter when used in conjunction with a variety of fluorescent reagents to detect cell viability, apoptosis, cytotoxicity, and transfection efficiency. The reusable slide option reduces consumption cost.
Designed specifically for Invitrogen EVOS imaging systems, the Invitrogen EVOS Onstage Incubator is an environmental chamber that enables precise control of temperature, humidity, and three gases for time-lapse imaging of live cells under both physiological and nonphysiological conditions.
The Invitrogen HCA Onstage Incubator for Thermo Scientific CellInsight HCA platforms allows precise control of temperature, humidity, and CO2 levels so that you may observe and measure biological activity and changes over time. Data gathered from longer-term imaging studies are the basis of quantitative analysis studies, especially when combined with Thermo Scientific HCS Studio Software for increased statistical power.
Tips
|
Step 1: Plan
Design your experiment with careful consideration of the tools and resources needed for each step.
Advantages
- Observe dynamic cellular processes as they happen
- Study and image several processes and functions simultaneously using multiplexed assays
- Study cellular structures in their native environment, resulting in more realistic results closer to in vivo scenarios
- Track cellular biomolecules and structures over time
- Observe interactions between cells
- Cellular enzymes and other cytosolic biomolecules remain in the cell
Considerations
- Must have a specific way to label your target with minimal toxicity – whether it is a molecule, a cellular function, or a cellular state
- Living cells are generally not permeable to large detection molecules such as antibodies
- Moving objects can be more difficult to keep in focus
- Certain techniques can be harmful to living cells
- Cells must be kept in their natural physiological state
Step 2: Culture
Maintain or grow your cells in optimum conditions.
Keeping cells alive and healthy during various experimental manipulations, detection, and imaging is no small task. The choice of medium is particularly important for time-lapse imaging and experiments where cells are exposed to ambient conditions for longer periods. For reliable results with live cells, it is essential that the cells be healthy and kept in an environment as close as possible to physiological temperature, pH, oxygen level, and other conditions.
Product highlights
These media and wash buffers are created specifically for live-cell imaging and detection. Employing them in your experiments can help you improve image clarity, reduce background fluorescence, and optimize cell viability.
- Thermo Scientific Nunc cell culture vessels with Nunclon Delta surface treatment have been validated with Gibco media to confirm consistent cell growth across multiple cell lines. It’s a proven combination for happy cells and happy scientists.
- Gibco extracellular matrices, scaffolds, and proteins provide in vivo–like morphology and physiologically relevant environments for more realistic cell biology and better intercellular interactions.
- Invitrogen Countess 3 FL Automated Cell Counter is an affordable and automated tool for checking the health of your cells quickly and objectively. It has reusable or disposable slide options.
TipsYou can improve image clarity, reduce background fluorescence, and optimize cell viability by using media and wash buffers created specifically for live-cell imaging and detection. See product selection guide |
Step 3: Label
Target cell structures, cell functions, and proteins of interest with selective dyes and stains.
The appropriate fluorophore (targeted fluorescent protein or small membrane-permeant reagent) should be used to monitor your target cellular structure or process. Additional fluorophores can be used to monitor multiple cellular structures and processes, but the excitation and emission spectra should be checked using the Fluorescence SpectraViewer or Stain-iT Cell Staining Simulator to ensure minimal spectra overlap. It is critical to avoid using too much fluorescent label because excessive fluorescent labeling can result in:
- Nonspecific staining with increased background signals
- Physiological artifacts and structural perturbations
- Cytotoxicity
- Spectral overlap
Note that:
- Live-cell structure reagents—help identify cellular components
- Live-cell function reagents—help identify cellular functions and processes
Product highlights
- Invitrogen CellLight reagents have proven to be the easiest to use for labeling specific structures in live cells. Targeted fluorescent proteins are introduced using the Invitrogen BacMam transduction system; no molecular biology techniques are required. Simply add the reagent to your cells, incubate overnight, and you’re ready to image in the morning.
- Invitrogen CellTracker reagents are a diverse reagent class used for labeling mammalian cells to view changes in morphology or location. These nontoxic fluorescent dyes are designed to freely pass through cell membranes into cells, where they are transformed into cell-impermeant reaction products. Incubating cells with a CellTracker reagent for 30 minutes will provide at least 72 hours of fluorescent signal (typically three to six generations).
- Invitrogen pHrodo indicators are fluorogenic dyes that dramatically increase in fluorescence as the pH of their surroundings becomes more acidic. When conjugated to dextrans, proteins, or other particles, pHrodo dyes can be used as highly specific sensors of endocytic and phagocytic internalization and lysosomal sequestration in live cells, offering a superior alternative to conjugates of other fluorescent dyes such as fluorescein and tetramethylrhodamine.
Tips
|
Step 4: Optimize
Minimize background and maintain photostability of fluorescence signals.
Signal-to-background ratio can be optimized by using reagents that reduce extracellular fluorescence and increase fluorophore photostability. It is important to image in media that have been specifically designed for maintaining cell health while reducing or eliminating background fluorescence in live-cell imaging experiments (see Table 1). The addition of a background suppressor compatible with live cells can also help reduce extracellular background fluorescence and eliminate the need for a wash step. ProLong Live Antifade Reagent can be applied to samples to reduce photobleaching of fluorophores, preventing signal loss with multiple or long exposures.
Table 1. Imaging media comparison.
| Reagent | Cell washing | Short-term imaging | Imaging up to 4 hours | Long-term imaging |
|---|---|---|---|---|
| Gibco PBS, pH 7.4 | | | ||
| Gibco FluoroBrite DMEM | | | | |
| Invitrogen Live Cell Imaging Solution | | | |
Product highlights
- Invitrogen BackDrop Background Suppressor is used when observing high background signal or weak fluorescence in the blue, green, or red channels.
- Gibco PBS, pH 7.4 is ideal for cell washing and short-term imaging where prolonged incubation is not required.
- Invitrogen Live Cell Imaging Solution is an optically clear solution used for imaging, dye loading, and wash steps. It helps keep cells healthy for up to 4 hours.
Live HeLa cells labeled with Tubulin Tracker Green dye and Tubulin Tracker Deep Red dye. Both labels show high off-cell background when the probe is left in the staining solution (left). Addition of BackDrop Background Suppressor greatly reduces extracellular background while leaving intracellular labeling unaffected (right), thus enabling a no-wash protocol for high-contrast imaging of tubulin in live cells.
The overall signal protection offered by ProLong Live reagentcompared to untreated samples is calculated based on the scan number where treated and untreated samples reach the EC50 value. The addition of ProLong Live reagent permitted 100% more captures with Invitrogen CellLight Mitochondria-RFP reagent.
After 120 exposures using a standard time-lapse imaging protocol, samples treated with ProLong Live reagent are >20% brighter than untreated cells, enabling more data collection time.
Tips
|
Step 5: Image
Live-cell imaging of dynamic processes requires active observation over time
Illumination and detection
To minimize phototoxicity, choose imaging systems that give you the greatest control of light sources. Try to minimize light intensity, exposure time, wavelength range, and amount of excitation energy for illuminating your cells while still generating a good signal with low background. Use the illumination that gives you the highest signal with the lowest level of fluorophore excitation. In some cases (particularly when you wish to image over a long period of time), it is advisable to sacrifice resolution by using shorter exposure times or lower magnification in exchange for healthier cells.
Live-cell imaging over longer periods of time can be challenging because the target may move out of focus during the course of the experiment. Many microscopes have autofocusing features that can help keep your target in focus longer and reduce focal drift. Additionally, maintaining cells at a constant temperature and keeping the volume of solution in the vessel constant will help with focal drift.
Environmental control
Many cells cannot tolerate deviations from their optimal temperature, osmolarity, pH, and humidity. Requirements vary depending on what experimental question you are asking. For example, experiments investigating cell growth and division may have a different set of requirements than experiments involving receptor activation and calcium accumulation. Some robust immortalized cell lines will tolerate being imaged or monitored for short periods of time without any environmental control. Conversely, for long-term imaging and detection studies, good results with both immortalized cells and primary cells typically require tightly controlled environmental parameters.
Click image to enlarge
A scratch wound in a culture of HDFn cells loaded with Invitrogen CellTracker Deep Red Dye. (A) The illuminated area was subjected to repeated illumination for 10 hours. Cells in this area show signs of phototoxicity (a loss of viability as cells were not able to grow into the wound). (B) Cells in the non-illuminated area show viable cell growth into the wound.
The top cell shows catastrophic blebbing of the cell membrane caused by excessive light exposure. Blebbing is a term used to describe membrane perturbation caused by toxicity. By contrast, the bottom cell remains relatively healthy and is not displaying aberrant morphology.
Product highlights
To avoid the pitfall of proceeding to the next step in your experiment with unhealthy cells, a quick check for cell health can be done on the Countess 3 FL Automated Cell Counter when used in conjunction with a variety of fluorescent reagents to detect cell viability, apoptosis, cytotoxicity, and transfection efficiency. The reusable slide option reduces consumption cost.
Designed specifically for Invitrogen EVOS imaging systems, the Invitrogen EVOS Onstage Incubator is an environmental chamber that enables precise control of temperature, humidity, and three gases for time-lapse imaging of live cells under both physiological and nonphysiological conditions.
The Invitrogen HCA Onstage Incubator for Thermo Scientific CellInsight HCA platforms allows precise control of temperature, humidity, and CO2 levels so that you may observe and measure biological activity and changes over time. Data gathered from longer-term imaging studies are the basis of quantitative analysis studies, especially when combined with Thermo Scientific HCS Studio Software for increased statistical power.
Tips
|
Featured webinar
Best practices: 5 steps to live-cell imaging
Whether you are new to live-cell imaging or you are an experienced researcher, this webinar will show you how to obtain publication-quality live-cell images while avoiding the frustration of wasted time and resources.
Examples of live-cell images and videos
Live HeLa cells labeled with Tubulin Tracker Deep Red and NucBlue Live ReadyProbes Reagent, enabling visualization of the microtubule cytoskeleton and nuclei in live cells, respectively. High resolution images were acquired using a confocal system.
HeLa cells labeled using Invitrogen Tubulin Tracker Green dye and Invitrogen MitoTracker Red CMXRos dye show superb multiplexing capability and uniformity of staining compared with overexpressed fluorescent protein fusions. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 20X Super Apochromat objective using GFP and Texas Red EVOS light cubes.
HeLa cells labeled using NucBlue Live ReadyProbes Reagent, Tubulin Tracker Green dye, and Invitrogen LysoTracker Deep Red dye show superb multiplexing capability and staining specificity. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 60X Super Apochromat Oil objective using DAPI, GFP, and Cy5 EVOS light cubes.
U2OS cells labeled using Invitrogen LysoTracker Blue DND22 dye, Invitrogen MitoTracker Green FM dye, and Tubulin Tracker Deep Red dye show superb multiplexing capability and staining specificity. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 60X Super Apochromat Oil objective using DAPI, GFP, and Cy5 EVOS light cubes.
HeLa cells labeled using NucBlue Live ReadyProbes Reagent, Tubulin Tracker Green, and CellLight Mitochondria-RFP show superb multiplexing capability and staining specificity. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 60X Super Apochromat Oil objective> using DAPI, GFP, and RFP EVOS light cubes.
HeLa cells labeled using NucBlue Live ReadyProbes Reagent, ER-Tracker Green, and Tubulin Tracker Deep Red show superb multiplexing capability and uniformity of staining compared with overexpressed fluorescent protein fusions. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 20X Super Apochromat objective using DAPI, GFP, and Cy5 EVOS light cubes.
U2OS cells labeled using NucBlue Live ReadyProbes Reagent, CellLight Actin-GFP, CellLight Mitochondria-RFP, and Tubulin Tracker Deep Red show superb multiplexing capability and staining specificity. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 60X Super Apochromat Oil objective> using DAPI, GFP, RFP, and Cy5 EVOS light cubes.
HeLa cells grown in MEM with 10% FBS were transduced with CellLight Mitochondria-RFP and CellLight Golgi-RFP. After 24 hours, nuclei were counter stained with NucBlue Live ReadyProbes Reagent. Image was captured on an EVOS FL Auto 2 Imaging System using a 60X Apo objective and DAPI, GFP and RFP LED light cubes.
Four-plex image of COS7 live cell. Tubulin is labeled with Invitrogen Tubulin Tracker Green dye, mitochondria are labeled with Invitrogen MitoTracker Orange dye, plasma membrane is labeled with Invitrogen CellMask Deep Red dye, and nucleus is stained blue with Hoechst 33342 reagent. Image was taken with a Zeiss ELYRA S.1 microscope using Structured Illumination Microscopy (SIM). Image is provided to Thermo Fisher Scientific courtesy of M. Sauer and L. Behringer-Pließ, Biocenter University Wuerzburg.
Four-plex image of U2OS live cells. Tubulin is labeled with Tubulin Tracker Green dye, mitochondria are labeled with MitoTracker Orange dye, plasma membrane is labeled with CellMask Deep Red dye, and nuclei are stained blue with Hoechst 33342 reagent. Image was taken with an ELYRA S.1 microscope using Structured Illumination Microscopy (SIM). Image is provided to Thermo Fisher Scientific courtesy of M. Sauer and L. Behringer-Pließ, Biocenter University Wuerzburg.
HeLa cells were transduced with Invitrogen CellLight Mitochondria-RFP red dye and CellLight Talin-GFP green dye for 24 hours, then labeled with Invitrogen NucBlue Live ReadyProbes Reagent-Hoechst 33342 for 15 minutes. For photobleach protection, cells were incubated with Invitrogen ProLong Live Antifade Reagent for 90 minutes before imaging on an Invitrogen EVOS cell imaging system.
Three-plex image of COS7 live cell. Tubulin is labeled with Tubulin Tracker Green dye, plasma membrane is labeled with CellMask Orange dye, and nucleus is stained blue with Hoechst 33342 reagent . Image was taken using an ELYRA S.1 microscope using Structured Illumination Microscopy (SIM). Image is provided to Thermo Fisher Scientific courtesy of M. Sauer and L. Behringer-Pließ, Biocenter University Wuerzburg.
HeLa cells were transduced with CellLight Mitochondria-RFP red dye and CellLight Talin-GFP green dye for 24 hours, then labeled with NucBlue Live ReadyProbes Reagent-Hoechst 33342 for 15 minutes. For photobleach protection, cells were incubated with ProLong Live Antifade Reagent for 90 minutes before imaging on an EVOS cell imaging system.
Video gallery for live-cell imaging
U2OS cells treated with 50 mM menadione. Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM) (Cat. No. T668) mitochondrial potential indicator and fades with oxidative stress as indicated with CellROX Green reagent (Cat. No. C10444).
Hippocampal neurons labeled with Tubulin Tracker Deep Red (Cat. No. T34076).
U2OS cells transfected with CellLight Tubulin-GFP (Cat. No. C10613); CellLight Actin-RFP (Cat. No. C10583).
Live-cell imaging resources
For additional information and data, please see the following posters:
Poster: New generation sensors for caspase activation and mitochondrial superoxide in live cell microscopy
Poster: New and improved cellular health evaluation of 2D and 3D cellular models using microplate reader assays
Poster: Hypoxia measurements in live and fixed cells using fluorescence microscopy and high-content imaging
Poster: Evaluation of Cellular Senescence through Fluorescence Characterization
*Incucyte® Live-Cell Analysis System is trademarked or registered trademarks of Essen BioScience. Incucyte, Essen BioScience, and all names of Essen BioScience products are registered trademarks and the property of Essen BioScience unless otherwise specified. Essen BioScience is a Sartorius Company. MuviCyte™ live-cell imaging system is trademarked or registered trademarked of PerkinElmer. THUNDER Imager Live Cell & 3D Assay and MICA Microhub Microscope are sold by Leica Microsystems CMS GmbH.
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