Novel Lipophilic Tracking Dyes for Monitoring Cell Proliferation

Abstract

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.

Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution

“…While all five have been shown to be able to track proliferation with varying degree of success in primary lymphocytes [22,25], we have noted from our data and that of other groups the overall ''quality'' of peak resolution with lipophilic dyes is particularly poor [11,26] even after sorting to reduce fluorescence spread [18]. Our method was developed to specifically evaluate how any candidate dye for fluorescence proliferation tracking by dye dilution would perform in vitro, with particular emphasis on situations where the limits for peak resolution have been exceeded due to the inherent heterogeneity of the cell type.…”

“…Dye labelling was conducted as outlined previously [16,20] and per the manufacturers recommendations [11,12]. Briefly 2Â staining concentrations of CTV (C34557, Life Technologies, Paisley, UK), CTFR (C34564, Life Technologies), EPD (65-0840-90, eBiosciences, San Diego, USA), PKH26 (MINI26-1KT, Sigma, St Louis, USA) and CVC (MINCLARET-1KT, Sigma) were made up in either protein-free PBS (for succinimidyl dyes) or diluent C (for lipophilic dyes, provided with dyes).…”

“…It now meant that there was a system that could cope with single cell heterogeneity and also allow for multiplexing with phenotypic surface markers and cytokines to look at ''proliferentiation'' [9]. There are two different classes of dyes; succinimidyl esters that bind to the amine groups of cellular proteins only becoming fluorescent after cleavage by intracellular esterases [10,11] and lipophilic dyes that label the cell membrane [11,12]. In each case the principle is highly elegant and simple, as the input population divides the initial fluorescence should be apportioned to each daughter cell so that individual rounds of division are represented in measurement space by sharp peaks with roughly half the intensity of the parental population.…”

Highly Sensitive Real-Time In Vivo Imaging of an Influenza Reporter Virus Reveals Dynamics of Replication and Spread

“…For example, previous experiments monitoring inflammatory responses to influenza virus infection via PET imaging could be coupled with PASTN to determine the impact of the viral load and the site of replication on the severity of the response (55). Similarly, recruitment of discrete immune cell populations to sites of infection can be monitored by infecting mice with PASTN and detecting immune cells that have been labeled ex vivo with tracking dyes (56) or by detecting fluorogenic probes that are selectively activated by specific immune cell populations (57). Alternatively, innate immune responses and viral replication could be measured concurrently by using PASTN to infect reporter mice that drive firefly luciferase from an NF-B or IFN-␤ promoter(s), as NLuc and firefly luciferase exclusively use different substrates (29,58,59).…”

Enhanced Cellular Uptake of Albumin-Based Lyophilisomes when Functionalized with Cell-Penetrating Peptide TAT in HeLa Cells

“…For FACS, cells were stained with the plasma membrane dye PKH26 (Sigma Aldrich, Missouri, USA) [30], [31]. One million cells were incubated in 2 µM PKH26 dye in 500 µl buffer (according to manufacturer's protocol) for 5 min.…”