Michael Olszowy, Ph.D., global marketing program manager for Molecular Probes™ at Invitrogen (www.invitrogen.com), covered a wide range of nonantibody alternative reagents for labeling cells.
One of Invitrogen’s new product offerings is the line of Vybrant® DyeCycle™ stains, which perform as an alternative to Hoechst 33342, binding quantitatively to DNA. Such dyes are important in flow cytometry for studies on the cell cycle as they reveal the relative amount of DNA in populations of cells that may be actively passing through the cell cycle.
In contrast to dyes like propidium iodide that can only penetrate dead cells, these new dyes permeate living cells in the presence of media components and other materials. Three different dye options fluoresce in the violet (440), green (530), and orange (585) ranges. These spectral properties allow accurate cell-cycle analysis in living cells on the most common laser used in flow cytometry analysis, the blue, argon 488 nm laser.
In addition, because the Vybrant DyeCycle dyes are less toxic compared to other cell-cycle labels, including DRAQ5, the dyes can also be used in a cell sorter to separate out particular populations where the cells can be regrown and studied further, according to Invitrogen.
Moreover, with one of the dyes in particular, Vybrant DyeCycle Violet dye, stem cell side population analysis may be performed using a less toxic dye on the less toxic violet laser, reports the company.
According to Dr. Olszowy, Invitrogen has developed another product designed to facilitate the measurement of DNA synthesis in cellular populations. It is a technique that allows researchers to finally move from the long-standing technology of growing cells in the presence of radioactive thymidine, a technique first developed in the 1960s.
The use of tritiated thymidine was superseded through the application of bromodeoxyuridine (BrdU), a thymidine analog, which could be detected with an antiBrdU antibody, but only after extensive manipulation of the sample. Now Invitrogen scientists have developed a more simplified approach that takes advantage of Click Chemistry.
The term Click Chemistry was coined by Barry Sharpless, Ph.D., of Scripps Institute, to describe a series of simple reactions by which larger chemical units can be linked covalently in a nonbiologic reaction that minimizes background staining.
Using EdU, a different thymidine analog that enters into a simple azide-alkyne cycloaddition (the classic click chemistry reaction), it is possible to add a fluorescent molecule to newly synthesized DNA in situ. Because the detection reagent is a small azido group attached to a fluorochrome (total MW=843 vs the 150K antibody/fluorochrome), this low molecular weight reagent diffuses easily to native DNA to find its target. In this fashion investigators can generate data on the dynamics of cellular DNA synthesis that is far more rapid, accurate, and precise than that produced through traditional methods, according to Invitrogen.
The assay, available as Click-it™ EdU Cell Proliferation assay, requires about two hours to complete. Without denaturation steps or harsh treatments, little manipulation is required as opposed to traditional protocols such as BrdU incorporation, which can take up to one and a half days.
Invitrogen has also made a significant foray into the world of nanotechnology, exploiting Qdot nanocrystals, which are highly fluorescent, nanometer-sized, inorganic semiconductor crystals. The size of the particle determines its emission and absorption spectra because in this size range (10—20 nm), the structures come to obey quantum laws, which dictate their properties and hence their spectra. This means that it is possible to engineer a vast range of colored particles.
“The current crop of Qdot nanocrystals are comparable to the best of the organic dyes in intensity,” says Dr. Olszowy, “and the narrow emission spectra of the Qdot nanocrystals result in low cross-talk as the Qdot reagents exhibit minimal spectral overlap and there is never a problem with photobleaching.”