Cell movement and location studies require specialized probes that are nontoxic to living cells and are available in a range of fluorescent colors to match instrument lasers and filters and to accommodate co-staining with antibodies or other cell analysis probes. Choose passively loaded dyes for short- and long-term tracking or fluorescent fusion proteins for cell tracking over 5 or more days.
The simplest processes for monitoring cell movement and location use tracking probes that pass through the membrane into the cell and become membrane-impermeant after loading. Ideal tracking dyes are passed to daughter cells for multiple generations but do not transfer to neighboring cells on contact. CellTracker™ dyes exhibit ideal properties—they are stable, nontoxic at working concentrations, well retained in cells, and brightly fluorescent at physiological pH. CellTracker™ dyes have a wide range of emission spectra for easy combination with antibody staining. |
CellTracker™ dyes are non-toxic at working concentrations. HeLa cells were plated in 96-well plates at a density of 2,500 cells per well. The next day the cells were loaded with CellTracker (at 1 uM) or DMSO in Live Cell Imaging Solution (LCIS) for 60 min at 37°C, then washed and imaged in LCIS. Cell viability was measured daily using the CyQUANT® Direct Cell Proliferation Assay reagent and analyzed using the EVOS® FL Auto Imaging System. No cytotoxic effects were observed in association with any CellTracker™ dye treatment.
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Longer-term tracking of cell movement and location requires probes that are more resistant to photobleaching and are retained through more cycles of cell division. Qtracker® probes provide intense, stable signals for long-term (6–10 generations) tracking. The eight distinct probe colors are resistant to photobleaching and provide intense punctate fluorescence within the cell, with different emissions from a single excitation for easy combination with antibody staining. Qtracker® probes have a wide range of emission spectra for easy combination with antibody staining. |
Multiplexing Qdot® probes, dyes, and fluorescent proteins. U2-OS cells were transduced with CellLight® Plasma Membrane-GFP (green), followed by labeling with Qtracker® 655 Cell Labeling Kit (magenta), and HCS NuclearMask™ Blue (blue). |
Transiently transduced cells express fluorescent fusion proteins at different locations within the cell for at least 5 days or up to 2 weeks in slow-growing populations. Labeled cells are easy to track, but not all cells within a population will express the fluorescent protein. CellLight® fluorescent proteins can be expressed in a variety of cell types including numerous transformed cell lines, primary cells, stem cells and neurons. |
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Cell morphology and image segmentation
Identifying cell boundaries is a key step in automated image acquisition and analysis for high-content screening (HCS) assays. Image segmentation involves separating objects of interest (such as cells) from background or other features not relevant to the analysis. It allows quantitation of morphological changes and items of interest in their cellular context, such as the translocation of biomarkers between different organelles. We offer HCS CellMasks™ for whole cell demarcation in a variety of wavelengths for multiplexing together with other structural and functional probes. |
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Short-term tracking requires rapid loading and typical retention for 3–6 generations or 72 hours.
Properties
| CellTracker™ dyes | |
|---|---|
| Method of use | 15 minutes to 1 hour of cell loading |
| Readout | Internalized probe gives bright fluorescence throughout the cell |
| Range | Tracking extends for 3–6 generations or 72 hours |
| Compatibility | Multiplex easily with antibody labeling after fixing |
| Signal-to-noise |  |
| Photostability | |
| Fluorescence microscopy | Yes |
| HCS | Yes |
| Bibliography | Citations |
Selection Guide
| CellTracker™ dye | Common filter set | Ex/Em (nm) | Quantity | Cat. No. | |
|---|---|---|---|---|---|
| CellTracker™ Blue CMAC (7-amino-4-Chloromethylcoumarin) | CMAC | DAPI | 353/466 | 5 mg | C2110 |
| CellTracker™ Blue CMF2HC (4-chloromethyl-6,8-difluoro-7-hydroxycoumarin) | Blue CMF2HC | DAPI | 371/464 | 5 mg | C12881 |
| CellTracker™ Violet BMQC (2,3,6,7-tetrahydro-9- bromomethyl-1H,5H-quinolizino( 9,1-gh)coumarin) | Violet BMQC | DAPI | 415/516 | 5 x 0.1 mg | C10094 |
| CellTracker™ Green CMFDA (5-chloromethylfluorescein diacetate) | Green CMFDA | FITC | 492/517 | 20 x 50 µg | C7025 |
| CellTracker™ Orange CMRA | Orange CMRA | TRITC | 548/576 | 20 x 50 µg | C34551 |
| CellTracker™ CM-DiI | CM-DiI | TRITC | 553/570 | 20 x 50 µg | C7000 |
| CellTracker™ Red CMTPX | CMTPX | Rhodamine | 577/602 | 20 x 50 µg | C34552 |
| CellTracker™ Deep Red | Deep Red | Cy®5 | 630/650 | 20 x 15 µg | C34565 |
Long-term tracking requires longer photostability and typical retention for 6–10 generations; loading time is less critical to experimental protocols.
Properties
| Qtracker® 525 Cell Labeling Kit | |
|---|---|
| Method of use | 1-hour cell loading using a targeting peptide |
| Readout | Internalized probe gives intense punctate fluorescence within the cell |
| Range | Tracking extends for 6–10 generations or 5–14 days |
| Fixablility | Image cells live or after fixing |
| Compatibility | Multiplex easily with antibody labeling |
| Signal-to-noise |  |
| Photostability | |
| Laser excitation | Excited optimally with UV up to 488 nm (also every wavelength below the emission wavelength) |
| Fluorescence microscopy | Yes |
| HCS | Yes |
| In vivo | Yes |
| Bibliography | Citations |
Selection Guide
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Common filter set
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Emission max (nm)
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Quantity
|
Cat. No. | |
|---|---|---|---|---|
| Qtracker® 525 Cell Labeling Kit | GFP or FITC | 525 | 1 kit | Q25041MP |
| Qtracker® 565 Cell Labeling Kit | 565 | 1 kit | Q25031MP | |
| Qtracker® 585 Cell Labeling Kit | TRITC-A | 585 | 1 kit | Q25011MP |
| Qtracker® 605 Cell Labeling Kit | 605 | 1 kit | Q25001MP | |
| Qtracker® 625 Cell Labeling Kit | Texas Red® | 625 | 1 kit | A10198 |
| Qtracker® 655 Cell Labeling Kit | 655 | 1 kit | Q25021MP | |
| Qtracker® 705 Cell Labeling Kit | Cy®5 | 705 | 1 kit | Q25061MP |
| Qtracker® 800 Cell Labeling Kit | Cy®7 | 800 | 1 kit | Q25071MP |
Transduction with fluorescent proteins provides long-term tracking, but since not all cells are transformed some automated techniques are not supported.
| Method of use | Transduction of cell line with fluorescent protein requires overnight incubation | ||
| Readout | Up to 90% of cells express fluorescent protein in respective cell compartments | ||
| Range | Expressed for ≥10 cell generations; 5 days to 2 weeks of tracking | ||
| Dye |
RFP
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GFP
|
GFP
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| Ex/Em (nm) |
555/584
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488/510
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488/510
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| Fixability |
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| Multiplexing |
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| Signal-to-noise |
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| Photostability |
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| Location |
Nucleus
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Cytoplasm
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| Fluorescence microscopy |
Yes
|
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| In vivo |
Yes
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| Bibliography |
Citations
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| Quantity |
1 mL
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1 mL
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1 mL
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| Cat. No. |
C10603
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C10602
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B10383
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| HCS CellMask™ Red | HCS CellMask™ Deep Red | ||||
|---|---|---|---|---|---|
| Readout |
Stains nucleus and cytoplasm for whole-cell demarcation
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| Common filter set |
DAPI
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FITC
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RFP
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Texas Red®
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Cy®5
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| Ex/Em (nm) |
346⁄442
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493/516
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556/572
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588⁄612
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650⁄665
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| Signal-to-noise ratio |
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|
|
| Photostability |
|
|
|
|
|
| Multiplexing |
Yes
|
Yes
|
Yes
|
Yes
|
Yes
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| Live cells |
No
|
No
|
No
|
No
|
No
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| Fixed cells |
Yes
|
Yes
|
Yes
|
Yes
|
Yes
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| Platform |
HCS
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HCS
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HCS
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HCS
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HCS
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| Protocol | High-content analysis | ||||
| Format |
250 µg/10 plates
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250 µg/10 plates
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250 µg/10 plates
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250 µg/10 plates
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250 µg/10 plates
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| Cat. No. | |||||
Resources |
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| Molecular Probes® Handbook |
BioProbes® Journal articles |