Choose From Fluorescent Dyes, Qdot® Nanocrystals, and Targeted Fluorescent Proteins

With applications in the study of cell migration, wound healing, and stem cell differentiation, Molecular Probes® fluorescent cell tracers are essential tools for following the movement of cells in complex environments. In addition, because many of the strategies used in normal cell migration are employed by tumor cells during the process of metastasis and accompanying neovascularization, these tracers provide a means of visualizing cell movements in the search for therapeutic approaches that block these pathways.

We have expanded our selection of fluorescent cell tracers far beyond the traditional calcein probes to include reactive fluorophores and intensely fluorescent nanocrystals, as well as genetically encoded fluorescent proteins (Table 1). Although there are unique features of each probe class, and some minor differences between the probes within a class, each of these probes is specifically designed to meet the requirements of a useful cell tracer. They are cell permeant (or expressed intracellularly, in the case of the fluorescent proteins), stable, and nontoxic at optimized working concentrations; well retained in cells during the experimental interval (from hours to days); and brightly fluorescent at physiological pH. Moreover, these cell tracers are available in fluorescence emission colors that span the spectrum, allowing them to be combined with other live-cell probes for simultaneous detection of cell movement and other critical cell functions.

Short-Term Cell Tracers: Calceins

The nonfixable calcein violet AM, calcein blue AM, calcein green AM, and calcein red-orange AM esters are proven tracers for viability studies (Table 1). These calcein probes contain acetoxymethyl (AM) moieties that block the inherent negative charges on the molecules, allowing their passive diffusion across cell membranes. Once inside the cell, nonspecific esterases cleave the AM esters to produce cell-impermeant polar molecules that can be efficiently retained in the cell for several hours. Unlike CellTracker™ and CellTrace™ probes described below, the calcein AM probes are retained only in live cells with esterase activity; they do not label dead or dying cells with compromised membranes. When paired with a cell-impermeant nucleic acid stain such as SYTOX® Green dye or propidium iodide, these calcein tracers are effective for differentiating viable and dead cells using fluorescence microscopy, flow cytometry, or microplate-based fluorometry.

Mid-Term Cell Tracers: CellTracker™ and CellTrace™ Reactive Probes

For applications that require following cells for a longer period, we offer CellTracker™ (thiol-reactive) and CellTrace™ (amine-reactive) fluorescent tracers (Table 1). The cell-permeant CellTracker™ probes are available with violet, blue, green, orange, or red fluorescence. Once inside a live cell, these mildly thiol-reactive chloromethyl derivatives undergo what is believed to be a glutathione S-transferase–mediated reaction to produce cell-impermeant dye adducts. When stained with CellTracker™ probes, many cell types are both fluorescent and viable for at least 24 hours after loading and often through several cell divisions. Not only are the CellTracker™ dyes better retained in live cells than the calceins (Figure 1), but they are also retained after formaldehyde-based fixation for downstream immunocytochemical analysis. Figure 2 shows the use of CellTracker™ Green CMFDA in a wound-healing assay in the absence and presence of cytochalasin D, which disrupts actin polymerization.

The CellTrace™ probes are also well retained in cells and are retained after formaldehyde-based fixation once they have reacted with proteins or other amine-containing biomolecules. These amine-reactive cell tracers—including CellTrace™ Violet, CellTrace™ CFSE, and CellTrace™ Far-Red DDAO-SE probes—each contain a succinimidyl ester that reacts with available primary amines located both inside and outside the cell. The Sloan lab and colleagues have used CellTrace™ and CellTracker™ probes extensively to visualize the migration of tumor cells through three-dimensional extracellular matrices [1]. The bright, homogeneous staining of CellTrace™ Violet probe shows very little fluorescence variation between cells in a population, allowing visualization of distinct generations of proliferating cells in a fluorescence histogram (Figure 3).

Cell retention of CellTracker™ and calcein probes  
Figure 1. Analysis of the cell retention of CellTracker™ and calcein probes.
Jurkat cells were labeled with 1.0 µM calcein green AM, fluorescein diacetate, CellTracker™ Green CMFDA , calcein red-orange AM, CellTracker™ Orange CMRA, or CellTracker™ Red CMTPX . An equal amount of cells from each sample was analyzed by flow cytometry every 2 hr.

CellTracker™ and calcein probes
Figure 2. Wound-healing assay with CellTracker™ Green CMFDA. (Left) After growing in culture for 72 hr, a monolayer of Gibco® human neonatal dermal fibroblasts was stained with CellTracker™ Green CMFDA. (Middle) The stained cell monolayer was scratched and left to heal for 24 hr. Cells have reinvaded the wound but are not as dense as in the control plate. (Right) The stained cell monolayer was scratched and then treated with 0.1 μM cytochalasin D for 24 hr to disrupt actin polymerization, resulting in no closure of the wound site.

CellTrace™ Violet stain  
Figure 3. Generational analysis using CellTrace™ Violet stain. Human peripheral blood lymphocytes were harvested and stained with CellTrace™ Violet stain. The violet peaks represent successive generations of cells stimulated with mouse anti–human CD3 antibody and interleukin-2, and grown in culture for 7 days. The peak outlined in black represents cells that were grown in culture for 7 days with no stimulus.

Long-Term Cell Tracers: Qtracker® Cell Labeling Kits

The Qtracker® Cell Labeling Kits—available with Qdot® nanocrystals in seven brilliant colors—contain the reagents needed to deliver fluorescent Qdot® nanocrystals into live cells, providing a powerful method for real-time tracking studies over extended experimental time periods. These Qdot® nanocrystals contain a targeting peptide that allows their selective uptake into the cell, where they are distributed in vesicles throughout the cytoplasm (Figure 4).

Inside the cell, the Qdot® nanocrystals exhibit an intense, photostable fluorescence that can be observed using continuous illumination, without time constraints due to photobleaching or degradation. Their fluorescence is maintained in complex cellular environments and under various biological conditions, including changes in intracellular pH, temperature, and metabolic activity, and they are passed to daughter cells through at least seven generations, or about 8 days in U2OS cells [2,3]. Moreover, the Qdot® nanocrystals are not transferred to adjacent cells in the population, making them useful for separating human embryonic stem (hES) cells from the mitotically inactive mouse embryonic fibroblast (MEF) feeder cells with which they are commonly co-cultured (Figure 5). Researchers have demonstrated a diversity of applications for cells labeled with the Qtracker® Cell Labeling Kits, including in vivo tracking of hematopoietic stem cells [4], following Schwann cells after injection into nerve grafts [5], and detecting neutrophil survival in Staphylococcus aureus–infected skin wounds [6]. Cells labeled with Qdot® nanocrystals are easily monitored on a variety of platforms, including traditional fluorescence microscopy, flow cytometry, fluorescence microplate readers, and high-content imaging systems. The cytotoxicity of the materials used in Qtracker® kits has been tested in many cell lines, including CHO, HeLa, U-118, 3T3, HUVEC, and Jurkat cells; labeling with these kits appears to exert minimal impact on cell-surface marker expression, cell proliferation, cellular enzyme activity, and cell motility.

Distribution of Qtracker® labels in cytoplasmic vesicles  
Figure 4. Distribution of Qtracker® labels in cytoplasmic vesicles. HeLa cells were labeled with the Qtracker® 655 Cell Labeling Kit. A Leica TCS SP2 confocal microscope was used to observe the Qdot® 655 nanocrystals (excitation at 488 nm).

hES cells co-cultured with mouse embryonic fibroblasts (MEF)
Figure 5. Immunocytochemical analysis of human embryonic stem (hES) cells co-cultured with mouse embryonic fibroblasts (MEF). BG1vp22 and SA2p12 hES cells were each plated onto a Qtracker® nanocrystal–labeled MEF feeder layer and allowed to grow for 48 hr. The cells were then fixed and labeled with anti-Oct4 or anti–Tra‑1‑81 primary antibody and green-fluorescent Alexa Fluor® 488 secondary antibodies. (Left) A colony of Oct4-expressing BG1vp22 cells (green) co-cultured with Qdot® 655 nanocrystal–labeled MEF (red) and counterstained with DAPI (blue). (Right) A suspension of Tra‑1‑81–expressing SA2p12 cells (green) co-cultured with Qdot® 655 nanocrystal–labeled MEF (red).

Long-Term Cell Tracers: CellLight® Reagents

CellLight® reagents are ready-to-use fusion constructs of emGFP and TagRFP for the expression of untargeted, cytoskeleton-targeted, or organelle-targeted fluorescent proteins. These prepackaged baculovirus reagents are powered by BacMam technology, which combines highly efficient gene delivery with a potent mammalian promoter for efficient transient expression of fluorescent proteins and prolonged visualization of cell structures. To achieve highly efficient expression—even in sensitive cells such as stem cells, neurons, and primary cells—CellLight® reagents are simply added to cells in complete medium. After incubation, the transduced cells are ready for imaging; no harsh transfection procedures or tedious cloning is required.

The CellLight® cytoplasmic marker—also called BacMam GFP transduction control—produces emGFP expression in the cytosol and nucleus of a wide range of cell types. The transiently transduced cells typically express the fluorescent protein fusion for about 5 days in standard cell lines (e.g., HeLa and CHO). With slowly dividing cells—such as some primary cells—expression has been demonstrated for up to two weeks, and with terminally differentiated neurons we have images recorded more than three weeks after transduction.

Table 1. Molecular Probes® fluorescent cell tracers.

Probe class

Selected Cat. Nos.


General attributes

Retention time

Instrument platform *

Calcein AM probes



Acetoxymethyl (AM) ester–modified compounds that can passively diffuse across cell membranes, where nonspecific esterases cleave the AM ester moieties to produce a product that is retained onlyin live cells.

<1 day (1–3 hr)


CellTracker™ thiol-reactive probes



Cell-permeant, thiol-reactive labeling reagents containing a chloromethyl moiety that can react with intracellular thiol groups from cysteine residues in proteins or glutathione.

72 hr or 3–4 generations


CellTrace™ amine-reactive probes



Cell-permeant, amine-reactive labeling reagents containing a succinimidyl ester that forms stable adducts with primary amines located intra- and extracellularly.

5–10 days or 8–10 generations


Qtracker® Cell Labeling Kits



Provides Qdot® nanocrystals that contain a selective targeting peptide for uptake into the cytoplasm of live cultured cells. Once inside cells, Qdot® nanocrystals provide intense, stable fluorescence that can be traced through several generations; they are not transferred to adjacent cells in a population.

Up to 1 week


CellLight® fluorescent protein–based reagents


Yes †

Baculovirus-packaged DNA constructs for the expression of nontargeted fluorescent proteins or of fluorescent proteins fused to signal peptides for specific targeting to key cytoskeletal structures and organelles.

Days to weeks


* FC = flow cytometry. FM = fluorescence microscopy. HCS = high-content screening and analysis. M = microplate assay. † After fixation, these emGFP and tagRFP fusions can be detected with anti-GFP antibodies and anti-RFP antibodies, respectively.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.