January 2008

Three publications describing applications of new CellVue® dyes appeared in the most recent volume of Immunological Investigations. References are provided below:

  • Gertner-Dardenne J, Poupot M, Gray B, Fournie JJ. Lipophilic Fluorochrome Trackers of Membrane Transfers between Immune Cells. Immunological Investigations, 36, No 5-6, 665-685 (2007)
  • Bantly AD, Gray BD, Breslin E, Weinstein EG, Muirhead KA, Ohlsson-Wilhelm BM, Moore JS. CellVue® Claret, a new Far-Red Dye, Facilitates Polychromatic Assessment of Immune Cell Proliferation. Immunological Investigations, 36, No 5-6, 581-605 (2007)
  • Tario, JD, Gray BD, Wallace SS, Muirhead KA, Ohlsson-Wilhelm BM, Wallace PK. Novel Lipophilic Tracking Dyes for Monitoring Cell Proliferation. Immunological Investigations, 36, No 5-6, 861-885 (2007)

The studies by Gertner-Dardenne et al. illustrate the use of CellVue Maroon labeled membranes for the study of cell to cell transfers of membrane molecules between lymphoid cells (also known as trogocytosis) using either flow cytometry or time-lapsed video-microscopy techniques. Interestingly, in these studies, the authors were able to simultaneously visualize trogocytosis, perforin release and cell death at the single cell level of a lytic immunological synapse as shown in the movie to the right.

The studies by Bantly et al. show that CellVue Claret can be used as a membrane label for T-lymphocyte proliferation studies using a BD FACS Caliber flow cytometer for cell analysis. It was reported that lymphocyte recovery, viability and surface immunophenotype are unaffected by labeling with this new far-red emitting dye. Therefore, this membrane labeling dye is a useful alternative to CFSE or PKH26 when the visible wavelength spectral channels are needed for other probes in multi-parametric flow cytometric studies.

The work by Tario et al. shows that CellVue Lavender, CellVue Plum and CellVue NIR780, are similar to established tracking dyes such as PKH26 and CFSE in terms of staining procedure, membrane stability, optimal concentration, and lack of effect on cellular proliferation. It was also shown that by using an asynchronously cycling population of U937 cells, which exhibits an approximately 24-hour doubling time, CellVue Plum, CellVue NIR780 and PKH26 are useful for discerning at least six divisions from the parental population, while CellVue Lavender and CFSE can be used to distinguish at least five. Since CellVue Lavender, CellVue Plum and CellVue NIR780 have fluorescence emissions in the violet, far-red and near infrared wavelength regions, respectively, and spectrally distinct from existing visible tracking probes, they can provide more detection alternatives when employed in cell labeling experiments. In particular, the CellVue dyes capitalize upon the expanding number of lasers incorporated into commercial instruments; thus allowing measurement of labeled cell populations in underexploited regions of the spectrum.

One publication describing a new NeuroVue dye, NeuroVue Jade, and its application for neuronal tracing studies also appeared in the most recent volume of Immunological Investigations

  • Jensen-Smith H, Gray B, Muirhead K, Ohlsson-Wilhelm, B and Fritzsch B. Long-Distance Three-Color Neuronal Tracing in Fixed Tissue Using NeuroVue Dyes. Immunological Investigations., 36, No 5-6, 763 (2007)

In this paper it is reported that NeuroVue Jade (NV Jade) is an improved version of the previous green fluorescent NV dye analogs, NV Emerald and NV Green, and is suitable for neuronal tract tracing studies in fixed tissue of up to at least five days. Typical diffusion rate of this dye in tissue is 1mm per day and it can be readily imaged in combination with NV Red and NV Maroon using either epifluorescence or confocal microscopy. Results also show that NV Jade can be used to visualize the very thin nerve fibers (0.2-0.5µm thick) in the brains of near-adult (18 day old) wild-type mice after a 5-day incubation at 37oC at distances up to 5mm away from the insertion site (Figure 1, Panel A). Images in Figure 1 (Panels B and C) also show that three-color imaging with NV Jade, NV Red and NV Maroon can be used to assess myelinated projections from three other regions of the 18 day old mouse brain.

nvjade

Figure 1: Three-color imaging with NV Jade, Red and Maroon allows excellent resolution of thin fibers and discrete nerve tracts in near-adult murine brain. Nerve tracts projecting from one region of mature brain to another were labeled with NV Jade (green pseudocolor), NV Red (red pseudocolor) or NV Maroon (blue pseudocolor) and imaged by confocal microscopy after 5 days of diffusion at 37oC. Bar indicates 100µm in all images. Panel A: Despite increased green autofluorescence due to myelinization of near-adult brain, NV Jade can be used in 18-day-old cerebella to label not only parallel fibers (green lines crossing from top left to bottom right) but also individual cells (stellar neuron in the center of A) at distances of up to 5mm away from the injection site. (Representative image selected from 12 replicate specimens). Panels B and C: After labeling in the pontine nuclei, cerebellar cortex and restiform body of the cerebellum with NV Jade, NV Red and NV Maroon, respectively, discrete fibers are consistently labeled and readily visualized near the deep cerebellar nuclei of P18 animals without bleed through and at distances of up to 1 cm away from the injection site over 50 animals. The same type of labeling was used on two replicate specimens, which were cut slightly differently to show fiber bundles running in parallel (B) or crossing one another (C). Reprinted by permission of Taylor and Francis Ltd. from Jensen-Smith paper cited above.