Features of the CellTracker dyes:
• Easy to use—remove culture media, add dye, incubate 15-45 minutes, and image cells
• Excellent retention—fluorescent signal retention of >72 hours (typically three to six generations)
• Ideal tracking dyes—monitor cell movement, location, proliferation, migration, chemotaxis, and invasion
• Low cytotoxicity—does not affect viability or proliferation
• Cell versatility–works with cells in suspension and adherent cells

The CellTracker fluorescent probes have been designed to freely pass through cell membranes; however, once inside the cell are transformed into cell-impermeant reaction products. The CellTracker Green and Red fluorescence probes contain a chloromethyl or bromomethyl group that reacts with thiol groups, utilizing a glutathione S-transferase–mediated reaction. In most cells, glutathione levels are high (up to 10 mM) and glutathione transferase is ubiquitous. The CellTracker Deep Red probe contains a succinimidyl ester reactive group, which reacts with amine groups present on proteins.

The strong, photostable fluorescence, excellent cellular retention, and minimal cytotoxicity of DiI and other long-chain carbocyanine membrane probes make them particularly suitable for long-term labeling and tracking of cells, intracellular membranes, liposomes, viruses, and lipoproteins. CellTracker CM-DiI incorporates a mildly thiol-reactive chloromethyl substituent that confers aldehyde fixability via conjugation to thiol-containing peptides and proteins. Unlike DiI and PKH26, CM-DiI is retained in cells throughout fixation, permeabilization, and paraffin embedding procedures.

After conversion to impermeant versions, the CellTracker fluorescent probes are well retained in living cells through several generations. The probes are transferred to daughter cells but are not transferred to adjacent cells in a population. Cells loaded with the CellTracker fluorescent probes display fluorescence for at least 72 hours and exhibit ideal tracking dye properties—they are stable, nontoxic at working concentrations, well retained in cells, and brightly fluorescent at physiological pH. Additionally, several CellTracker fluorescent probes with various excitation and emission spectra are available allowing for multiplexing in fluorescence imaging. The CellTracker dyes are retained with fixation and permeabilization, enabling their use with antibodies for fluorescence immunostaining applications. In addition to bright fluorescence and excellent retention, the CellTracker fluorescent probes do not contribute to cytotoxicity.

Spectral characteristics of the fluorescent CellTracker probes:
• CellTracker Green CMFDA—492/517 nm
• CellTracker Red CMTPX—577/602 nm
• CellTracker Deep Red—630/660 nm
• CellTracker CM-DiI Dye—553/570 nm

The fluorescent CellTracker reagents include the green-fluorescent chloromethyl derivatives of fluorescein diacetate (CMFDA), red-fluorescent CMTPX and CM-DiI, and Deep Red. CellTracker Red CMTPX does not require enzymatic cleavage to activate its fluorescence, whereas the green CMFDA does require enzymatic cleavage. CMFDA is colorless and non-fluorescent until cytosolic esterases cleave off the acetates, releasing a brightly fluorescent product.