Cell Tracking

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.

Select products for cell tracking

Short-term cell tracking

  • Rapid loading (typically 15 min to 1 hr)
  • Short-term tracking (typically 3–6 generations)
  • Moderate resistance to photobleaching
  • Moderate brightness
  • Signal distribution throughout the cell

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.

 

Enlarge image

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.

Long-term cell tracking

  • Typically 1-hour cell loading
  • Long-term tracking (typically 6–10 generations)
  • High resistance to photobleaching
  • Intense brightness
  • Punctate distribution throughout the cell

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.

 

Long-term cell tracking

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).

Cell tracking with fluorescent proteins

  • Simple transduction protocol
  • Overnight incubation
  • Long-term tracking for up to 2 weeks or more than 10 generations
  • Red or green fluorescent protein
  • Can be multiplexed with fluorescent dyes or proteins

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.

 

Cell tracking with fluorescent proteins

Porcine primary skeletal muscle cell transduced with CellLight® Nucleus-GFP fluorescent proteins.

Cell morphology and image segmentation

  • Label nucleus and cytoplasm
  • No wash steps required
  • Multiple wavelength options
  • Validated for automated imaging

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.

 

HeLa cells labeled to indicate new DNA synthesis

HeLa cells were labeled with Alexa Fluor® 488 Click-iT® Plus EdU to indicate new DNA synthesis. β-catenin is stained using an anti-β-catenin antibody followed by an Alexa Fluor® 647 goat anti-mouse IgG secondary. Cell demarcation was achieved using HCS CellMask™ Blue stain, and the image was acquired using a CellInsight™ HCS instrument.

Cell tracking selection guides

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 3 stars
Photostability 3 stars
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 5 stars
Photostability 5 stars
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

 
Common filter set
Emission max (nm)
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
GFP
GFP
Ex/Em (nm)
555/584
488/510
488/510
Fixability
5 stars
Multiplexing
5 stars
Signal-to-noise
5 stars
Photostability
5 stars
Location
Nucleus
Cytoplasm
Fluorescence microscopy
Yes
In vivo
Yes
Bibliography
Citations
Quantity
1 mL
1 mL
1 mL
Cat. No.
C10603
C10602
B10383
  HCS CellMask™ Red HCS CellMask™ Deep Red
Readout
Stains nucleus and cytoplasm for whole-cell demarcation
Common filter set
DAPI
FITC
RFP
Texas Red®
Cy®5
Ex/Em (nm)
346⁄442
493/516
556/572
588⁄612
650⁄665
Signal-to-noise ratio
Photostability
Multiplexing
Yes
Yes
Yes
Yes
Yes
Live cells
No
No
No
No
No
Fixed cells
Yes
Yes
Yes
Yes
Yes
Platform
HCS
HCS
HCS
HCS
HCS
Format
250 µg/10 plates
250 µg/10 plates
250 µg/10 plates
250 µg/10 plates
250 µg/10 plates
Cat. No.