Betsy M. Ohlsson-Wilhelm

Diffusion and imaging properties of three new lipophilic tracers, NeuroVue ™ Maroon, NeuroVue ™ Red and NeuroVue ™ Green and their use for double and triple labeling of neuronal profile.

We describe here diffusion and imaging properties of three new lipophilic tracers, NeuroVue Maroon (near infrared), NeuroVue Red and NeuroVue Green. Using pair-wise comparisons between the new dyes and existing dyes (DiI, DiA, DiD, DiO, PKH2, PKH26) applied to the left and the right side of fixed spinal cord preparations, we show that NeuroVue Maroon (excitation maximum 647 nm) surpasses all other dyes in this study in signal to noise ratio. We also present data showing the utility of these new dyes for both double labeling and triple labeling in combination with each other or existing lipophilic tracers. Using mice bearing the PLP-eGFP transgene, we demonstrate that either NeuroVue Maroon or NeuroVue Red can readily be combined with eGFP labeling. Double labeling experiments using NeuroVue Red and eGFP allowed us to demonstrate that every fiber in the neonatal ear is surrounded by developing Schwann cells.

Long-distance three-color neuronal tracing in fixed tissue using NeuroVue dyes.

Dissecting development of neuronal connections is critical for understanding neuronal function in both normal and diseased states. Charting the development of the multitude of connections is a monumental task, since a given neuron typically receives hundreds of convergent inputs from other neurons and provides divergent outputs for hundreds of other neurons. Although progress is being made utilizing various mutants and/or genetic constructs expressing fluorescent proteins like GFP, substantial work remains before a database documenting the development and final location of the neuronal pathways in an adult animal is completed. The vast majority of developing neurons cannot be specifically labeled with antibodies and making specific GFP-expressing constructs to tag each of them is an overwhelming task. Fortunately, fluorescent lipophilic dyes have emerged as very useful tools to systematically compare changes in neuronal networks between wild-type and mutant mice. These dyes diffuse laterally along nerve cell membranes in fixed preparations, allowing tracing of the position of a given neuron within the neuronal network in murine mutants fixed at various stages of development. Until recently, however, most evaluations have been limited to one, or at most, two color analyses. We have previously reported three color neuronal profiling using the novel lipophilic dyes NeuroVue (NV) Green, Red and Maroon (Fritzsch et al., Brain. Res. Bull. 66: 249–258, 2005). Unfortunately such three color experiments have been limited by the fact that NV Green and its brighter successor, NV Emerald, both exhibit substantially decreased signal intensities when times greater than 48 hours at 37°C are required to achieve neuronal profile filling (unpublished observations). Here we describe a standardized test system developed to allow comparison of candidate dyes and its use to evaluate a series of 488 nm-excited green-emitting lipophilic dyes. The best of these, NV Jade, has spectral properties well matched to NV Red and NV Maroon, better solubility in DMF than DiO or DiA, improved thermostability compared with NV Emerald, and the ability to fill neuronal profiles at rates of 1 mm per day for periods of at least 5 days. Use of NV Jade in combination with NV Red and NV Maroon substantially improves the efficiency of connectional analysis in complex mutants and transgenic models where limited numbers of specimens are available.

Use of PKH Membrane Intercalating Dyes to Monitor Cell Trafficking and Function

Since their introduction as improved agents for in vitro and in vivo cell tracking,34,56,79 the lipophilic membrane intercalating fluorochromes known as PKH dyes have been used to study a wide variety of cell types and biological processes. Originally developed by Horan and colleagues at Zynaxis Cell Science as part of a larger class of multifunctional drug delivery molecules known as Zyn-Linkers, this family of patented fluorescent dyes and cell labeling reagents became available in kit form for research use in 1989 and was purchased by Phanos Technologies in 1995. PKH dyes are therefore found in the literature under a variety of names, including Cell Linker dyes, Zyn-linkers, Zyn-linker® dyes, cell tracking dyes, and PKH dyes. A number of suppliers are also found in the literature, including Zynaxis Cell Science (which no longer sells these reagents), Sigma and Dainippon Pharmaceuticals Laboratory Products Division (both of which continue to distribute PKH kits for Phanos).

Novel Lipophilic Tracking Dyes for Monitoring Cell Proliferation

The advent of contemporary digital instrumentation has enhanced both the potential and the complexity of flow cytometric experiments, allowing for the detailed dissection of immune cell subsets and their functions. The use of cell tracking labels such as PKH26 and CFSE has been important in observing such cellular functions, but their visible emission characteristics have limited the design of such analyses. As the demand for multiparametric flow cytometry intensifies, it will become increasingly important to utilize a broader range of cell tracking reagents to optimize the measurement of fluorescence signals and to provide flexibility in the use of commercially available fluorochrome - antibody combinations. We report on the evaluation of three lipophilic membrane dyes, CellVue® Lavender, CellVue® Plum and CellVue® NIR780; with fluorescence emissions in the violet, far-red and near infrared wavelength regions, respectively. These reagents 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. The CellVue dyes however, exhibit different spectral characteristics than existing tracking compounds, and capitalize upon the increased number of lasers incorporated into commercially available instrumentation; thus permitting measurement of labeled populations in underexploited regions of the spectrum.

CellVue ® Claret, a New Far-Red Dye, Facilitates Polychromatic Assessment of Immune Cell Proliferation

Flow cytometric analyses of immune cell proliferation, differentiation, and function are limited by the number of different fluorochromes that can be resolved simultaneously. Additional colors to expand functional analytic capability will facilitate higher dimensional analyses of heterogeneous cell populations by basic and clinical scientists. Our aim in these studies was to evaluate CellVue® Claret, a fluorescent, far-red emitting, membrane intercalating dye (excitation maximum: 655 nm, emission maximum 677nm), as an alternative and/or complementary probe to PKH26 and CFSE1 for polychromatic studies of immune cell proliferation and function. Using a BD FACSCalibur and human peripheral blood mononuclear cells (PBMCs) from 8 different donors (2 donors studied twice), we compared CellVue® Claret with the two most commonly used visible-emitting proliferation dyes, PKH26 and CFSE, in terms of: (1) compatibility with 7-Amino-actinomycin D (7-AAD) as a viability marker; (2) effect of dye labeling on lymphocyte viability; and (3) the proliferative response of CD3+ T lymphocytes from 0–96 hours as assessed by dilution of each of the 3 cell tracking dyes in cultures stimulated with anti-CD3 plus IL-2. Post-labeling recoveries and viabilities were similar for all 3 dyes, with modestly higher initial staining intensities and coefficients of variation for CellVue® Claret than for CFSE or PKH26. Lymphocyte viabilities in stimulated or unstimulated cultures were also unaffected by choice of dye. Proliferative responses of viable CD3+ lymphocytes were comparable for all three dyes, whether results were reported as Proliferative Fraction (percent of cells that had divided one or more times) or as Precursor Frequency (percent of parent population that had gone on to proliferate in response to anti-CD3 plus IL-2). In summary, T cell proliferation analysis using CellVue® Claret gives results equivalent to those obtained with PKH26 or CFSE, expanding the choice of proliferation dyes suitable for use in high dimensional polychromatic studies on flow cytometers with far red (633 nm–658 nm) excitation capabilities.

Zyn-Linked colchicines: Controlled-release lipophilic prodrugs with enhanced antitumor efficacy

Abstract Conjugation of antimitotic colchicine derivatives with proprietary lipophilic molecules (Zyn-Linkers™) via acid cleavable linkages (hydrazone or imine) produced prodrugs with enhanced antitumor activity. The pharmacodynamic properties (half-lives) of the pH-sensitive linkages were determined around values which might be expected in intracellular, especially lysosomal, environments. In buffered solutions at pH 4.1, 5.6, and 7.2, conjugates released 50% of the drug at rates from 0.1 h at pH 4.1 to over 3 months at neutral pH. In a tumor cell cytotoxicity assay, the slowest releasing molecules were up to 100-fold less active in vitro than the unlinked drug, which demonstrated that the linkages were stable and that a true prodrug had been generated. Binding of conjugate to cells, release of active drug and action on an intracellular target (tubulin) were demonstrated by the ability of the conjugates to block cells in the G2/M phase of the cell cycle 20 h after a 10 min exposure and was consistent with the rate of drug released. In vivo, a single injection of the Zyn-Linked conjugated colchicines enhanced the survival time of the mice to a greater extent than single doses of the unlinked colchicine analogues in a murine tumor model. Activity of the slowest releasing conjugate (ZYN 162) was shown to be comparable to a single LD 10 dose of doxorubicin. These data suggest that the Zyn-Linker conjugates described containing acid cleavable bonds form a body depot of prodrug which release an anti-mitotic agent and can significantly inhibit tumor growth.

Increased depth of cellular imaging in the intact lung using far-red and near-infrared fluorescent probes.

Scattering of shorter-wavelength visible light limits the fluorescence imaging depth of thick specimens such as whole organs. In this study, we report the use of four newly synthesized near-infrared and far-red fluorescence probes (excitation/emission, in nm: 644/670; 683/707; 786/814; 824/834) to image tumor cells in the subpleural vasculature of the intact rat lungs. Transpelural imaging of tumor cells labeled with long-wavelength probes and expressing green fluorescent protein (GFP; excitation/emission 488/507 nm) was done in the intact rat lung after perfusate administration or intravenous injection. Our results show that the average optimum imaging depth for the long-wavelength probes is higher (27.8 ± 0.7  μm) than for GFP (20 ± 0.5  μm; p = 0.008; n = 50), corresponding to a 40% increase in the volume of tissue accessible for high-resolution imaging. The maximum depth of cell visualization was significantly improved with the novel dyes (36.4 ± 1  μm from the pleural surface) compared with GFP (30.1 ± 0.5  μm; p = 0.01; n = 50). Stable binding of the long-wavelength vital dyes to the plasma membrane also permitted in vivo tracking of injected tumor cells in the pulmonary vasculature. These probes offer a significant improvement in the imaging quality of in situ biological processes in the deeper regions of intact lungs.