Cell Tracing and Tracking Support—Getting Started

This is not recommended. When these stains bind to DNA and RNA, they affect the normal function of the nucleic acids, disrupting transcription, as well as proliferation. Other reagents, such as CellTracker™ dyes or Qtracker™ reagents are more optimized for tracking without seriously disrupting normal activity. If a nuclear label is still desired, though, and the cells are mammalian and non-blood derived, CellLight™ nuclear reagents can transiently transfect cells to express GFP or RFP on a nuclear-expressing protein for up to several days without affecting function.

Calcein dyes diffuse into cells, the ‘AM’ moiety is cleaved by cellular esterases and then are observed in the cytoplasm without binding to anything. This means that they give a “whole cell” label. But it also means that they may be pumped out by normal cellular efflux mechanisms, sometimes within a very short time, especially for cell types that may exhibit drug resistance, unless the efflux is inhibited (such as with probenecid). It also means that the dyes are not crosslinked with aldehyde-based fixation, unlike protein-binding CellTracker™ dyes, and thus will be lost with fixation. Additionally, any disruption of plasma membrane, such as with detergents or trypsinization, will lead to leakage of the dyes from the cell.

Qtracker™ cell labeling reagents have a long publication history for this sort of usage. Being Qdot™ probes, they are bright enough to be detected over background despite multiple cell proliferations, even in tissue. They are non-transferrable between cells, unlike lipophilic cyanine dyes. And Qtracker™ cell labeling reagents are retained after fixation and permeabilization. They have been shown even to survive the heat and solvents of paraffin tissue processing, unlike the other cell tracking reagents.

Lipophilic cyanine dyes are preferred for this sort of assay, since they insert into cellular membranes and then, upon fusion, are shared by the fused cells as the membranes are shared. For example, one cell population can be labeled with DiI (orange-red) and another cell population can be labeled with DiO (green), and when the cells fuse, the combined color appears yellow (when imaged with a dual-bandpass filter set).

With excitation and emission maxima of 630/650 nm, any standard Cy5 or Alexa Fluor 647 filter set may be used. The reagent is efficiently excited using a 633 or 635 nm laser.

The CellTracker reagents have a reactive group that will allow covalent binding to various cellular components, large and small. When bound to small cellular components, if these are not cross-linked upon fixation, they may leach out, hence a loss of some signal.

The retention of signal is not based on time, but rather on the rate of cell division over time and, for some cell types, on the rate of active efflux, protein turnover or other metabolic events. Dependent upon the cell type, it may be possible to see signal that lasts far beyond 72 hours. On the other hand, it may also disappear sooner than 72 hours.

Degree of labeling (DOL) describes the number of fluorophores per antibody. For in vivo labeling experiments, the DOL is restricted to a narrow range because it has significant consequences for the biodistribution and clearance of the probe. We have determined that the DOL range for the far-red Alexa Fluor™ dyes is 1.5 to 3 molecules per antibody for optimal optical in vivo imaging. 

Antibodies should be at a concentration of 1.0–3.0 mg/mL. The antibody must be free of preservatives (azide), amine-containing buffers, and carrier proteins such as BSA.

Due to light scattering caused by fur, hairless mice such as athymic nude (nu/nu) mice are recommended for in vivo imaging. If this is not an option, the hair covering the area to be imaged should be removed using clippers or a chemical depilatory such as Nair™ depilatory. 

The volume of reagent that can be injected varies according to the route of administration. The following numbers are general guidelines for a 25 gram animal:

We recommend the use of a 28–32 gauge tuberculin or insulin syringe (0.3 or 1.0 mL volume) with a fixed (non-removable) needle.

A recommended starting dosage is 25–50 µL of Qtracker™ reagent diluted to the desired injection volume with PBS or normal saline. Qtracker™ reagent should be diluted immediately prior to injection. DO NOT STORE DILUTED. You will need to determine the optimal dosage for your experimental models.

A recommended starting dosage is 50 µg. You will need to determine the optimal dosage for your experimental model.

Quantum dots can be excited by wavelengths of light ranging from 250 nm to 40 nm below the emission peak wavelength. For example, Qtracker™ 655 non-targeted quantum dots can be excited from 250–615 nm. Remember that the wavelength in the product name refers to the emission peak, not the excitation peak. However, since quantum dots have an exponential curve to their absorbance, the lower the wavelength at which they are excited, the more efficiently they will absorb, so you will want to use your lowest available laser line or excitation wavelength.

The dye will be eliminated via the bladder. The bladder signal is detectable within 3 minutes of IV injection of the dye.

The imaging time course varies with the nature of the injected agent. Vascular tracers are visible in the blood vessels immediately after injection and may be imaged for several hours. Conjugated whole IgG antibodies reach their targets within a few hours of injection and may be imaged for several days.