Molecular Probes has a multitude of fluorescent dyes to selectively illuminate different organelles and membranes in many cell types (such as endoplasmic reticulum, Golgi, mitochondria, nucleus, endosomes and lysosomes). These fluorescent dyes work either by concentrating in a particular organelle or by exhibiting enhanced fluorescence in a certain cellular region or membrane. Organelle specific fluorescent probes have several uses; I have counter- and co-stained organelles, as opposed to using organelle specific antibodies, and I have also used the dyes for biophysical characterizations (for example, to investigate ER membrane continuity and fluidity with photobleaching techniques).
Most Molecular Probes organelle markers are simple to use and ‘flexible’. Selective labeling is generally achieved by simply adding the probe to cells (in ethanol or DMSO), incubating for an appropriate time and then washing away the excess. Further, many of the dyes can be used in live or fixed cells and have functional groups so that they are retained upon fixation if live cells were labeled. The Molecular Probes website (www.probes.com) and catalogue are very informative and facilitate the selection of appropriate fluorescent markers for a particular application. One region of mammalian cells that I have never had good results for with an organic probe was the plasma membrane. The available dyes I tried never produced a good stain and I was not willing to go to the effort of making liposomes to deliver dye. As a consequence, I tried Molecular Probes fluorescently labeled wheat germ agglutinin.
Wheat germ agglutinin (WGA) is a carbohydrate-binding protein of approx. 36 kDa that selectively recognizes sialic acid and N-acetylglucosaminyl sugar residues which are predominantly found on the plasma membrane. Molecular Probes produces WGA conjugated to fluorophores, including the Alexa Fluor fluorophores that have excellent photostability and illumination, are pH insensitive and may be used with a wide spectrum of emission wavelengths. Plasma membrane labeling with fluorescent WGA is very simple; cells are incubated at 1–10 g/ml WGA in standard buffers (PBS, HBSS) for 10 minutes and then washed. I have observed good labeling with low concentrations of WGA, thus the 5 mg package will label many samples. Both fixed and live cells can be labeled using the same protocol, although fixed samples should not be permeabilized and it should be noted that WGA is retained after fixation of live cells. As with many conjugated proteins, storage of aliquots at –20°C is recommended, as is centrifugation to remove any aggregates that may form.
I have used fluorescent WGA as a counter stain to delineate cells in fluorescent imaging and I have been very happy with the selectivity and brightness of staining. The large spectral range of fluorescent WGAs facilitates the use of this marker, in conjuction with other staining, for any microscopy work where the plasma membrane needs to be shown. Delineation of the plasma membrane in fluorescent images can be important particularly to highlight polarization of cellular factors in response to stimuli, to observe cellular projections during migration or wound healing assays or simply to show where your cells are. Other applications of WGAs include studies of isolated nuclei (as nuclear pore complexes include proteins that express N-acetylglucosaminyl residues), flow cytometry, identification of glycoproteins on gels and identification of fungi and gram-positive bacteria. Unfortunately, there are some down sides to using WGA with mammalian cells. Agglutinin can cause clustering of glycoproteins that can stimulate certain cellular pathways; this certainly needs to be considered before using this marker. Further, flatter cells give less distinct staining, although this is a problem intrinsic to the cell not the dye.
In conclusion, this probe achieves selective and simple staining of the plasma membrane, such staining can be very informative if you need to observe any change in membrane structure during a process such as migration or chemo-sensing. Unfortunately, activation of cellular pathways may limit the potential uses of this dye in dynamic/live cell imaging and other strategies to label the membrane may be necessary.
Peter Haggie, Ph.D.
Post-Doctoral Fellow
University of California, San Francisco