Product Survey: Cell labelling and tracking kits

Shine a Light
by Harald Zähringer, Labtimes 07/2013

Zebrafish larva with GFP labelled neurons.

Seeing is believing. That old saying is especially true for cell imaging.

Visualising cell components, organelles, whole cells, tissues, organs or complete organisms may reveal more about cell migration patterns or biological processes inside a cell than countless cell assays. To follow these processes with microscopes, flow cytometers or high content imagers, however, the cells or particular cell structures have to be labelled to render them visible for the imaging system.

Cell labelling or tracking vehicles found in corresponding kits may be roughly divided into two categories: endogenous reporter genes, which express fluorescent or other signal-generating proteins after cell transfer; and exogenous probes, such as fluorescent labelling and tracking dyes, that are passively taken up by the cells or are internalised via selective transport systems.

The green fluorescent protein (GFP) from the jelly fish Aquorea victorea and its blue, cyan and yellow fluorescent varieties are amongst the most popular reporter genes or proteins, respectively. There is hardly a cell or model organism that hasn’t been labelled with a GFP to visualise a single cell feature, a particular tissue structure or a whole organism. The GFP-colour palette has been completed in recent years by red fluorescent proteins from marine anemones and reef corals, and now comprises more than fifty fluorescent variants.

Not only GFP and luciferases

Reporter genes expressing luciferases from the American firefly Photinus pyralis, the sea pansy Renilla reniformis or the marine copepod Gaussia princeps are popular tools to label cells with a bioluminescence marker. Upon oxidation of their substrate (D-luciferin for firefly and coelenterazine for Renilla and Gaussia luciferase), the expressed luciferase produces a greenish (firefly) or blue (Renilla, Gaussia) luminescent light that may be detected with an adequate imaging system.

Though GFP and luciferase are almost the exclusive reporters used in biological labs, two other reporter-systems, namely PET and MRI reporters, are also common in clinical research to track, for example, transplanted stem cells. PET reporters are employed in Positron Emission Tomography (PET) to visualise cellular processes in organs, tumours, tissues or whole bodies. During a PET-scan a cyclotron generates positrons from a radionuclide, or PET-probe, that has been introduced into the cells. After clashing with an electron, the energy of the positron is transferred into two gamma rays. A ring-shaped PET-camera detects the radiation and computes an image showing the spatial distribution of the PET-probes inside the cells. So what’s the role of the PET-reporter in this process? The PET-reporter, usually an enzyme, receptor or transporter, e.g., thymidine kinase from the herpes simplex virus type I, acts as a PET-probe that assembles in a certain area of the cells and may be registered by the PET-camera.

Magnetic spin doctors

Magnet resonance imaging (MRI) is based on the nuclear magnetic resonance (NMR) of hydrogen nuclei, or protons, with radio waves. The spin of each proton, which may be thought of as a tiny magnet, interacts with the electromagnetic field of the radio waves and produces an NMR signal. Since human or animal bodies are basically a conglomerate of fat and water, the spin of myriads of protons adds to a strong signal that may be converted into an MR image.

Usually, a contrast agent that affects the proton spin is injected to enhance the MR signal intensity between two adjoining tissues. That’s where MRI reporter genes come into play. Typical contrast agents are ferromagnetic or paramagnetic materials such as iron nanoparticles or iron oxide. Hence, MRI reporters often express metalloproteins like transferrin and ferritin that accumulate iron ions intra­cellularly, to create a specific MR signal.

Usually, reporter genes are constructed to express only one of the above-mentioned reporter protein classes. However, they may also be combined to generate reporter genes for multimodal imaging. Johanna Niers and her colleagues from the Massachusetts General Hospital, the Harvard Medical School and the Cancer Center in Amsterdam recently presented a reporter construct armed with reporter genes for bioluminescence, fluorescence, PET and MR imaging (J. Am. Chem. Soc. 2012, 134, 5149-56). The all-purpose reporter is based on a vector harbouring a Gaussia luciferase (Gluc)-GFP fusion separated by an internal ribosome entry site.

Multi-armed reporter

The smart parts of the construct, however, are a biotin acceptor peptide (BAP) sequence and a transmembrane domain linked to the Gluc sequence. After expression, biotin ligase adds a biotin moiety to the BAP-domain of the Gluc-GFP fusion protein. The fusion is then transported to the cell surface, due to the natural secretion feature of Gluc and gets anchored to the membrane via the transmembrane domain. The rest is more or less routine. ­Added Streptavidin-coupled-PET-probes, -fluorophores or -magnetic particles bind to biotin for PET, fluorescence or MR imaging while the Gluc-domain may be utilised for bioluminescence imaging.

If cell labelling or tracking with reporter genes is not effective or even impossible, e.g., due to cell transfection issues, gene-transfer free labelling approaches based on dyes get their chance. Researchers may choose between countless types of labelling dyes with different chemical structures and characteristics, signal-generating principles, quantum yields, brightness or stability. The endless list contains organic and inorganic dyes, fluorescent dyes, quantum dots and nanoparticles, to name just the most common types.

Dyes may also be classified based on the labelled cell compartment into cytoplasmic, nuclear and membrane dyes. Amine and thiol reactive dyes, for example, are typical cytoplasmic dyes while lipophilic carbocyanine fluorescent dyes, such as the popular cell tracking dyes DiD, DiO and DiR bind to the cell membrane. Finding the best dye for a certain cell-labelling experiment seems harder than the experiment itself; however, you may find some orientation in the cell labelling and tracking dye jungle on the next pages.

First published in Labtimes 07/2013. We give no guarantee and assume no liability for article and PDF-download.

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