Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Abstract

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo. [1][2][3][4][5] Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:1. stem and progenitor cell quiescence, proliferation and/or differentiation [6][7][8] 2. antigen-driven membrane transfer 9 and/or precursor cell proliferation 3,4,10-18 and 3. immune regulatory and effector cell function 1,[18][19][20][21] .Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes 1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins 4,16,18 . Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class [2][3][4]16,18,24 .The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:1. Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies. 2. Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 10 2 -10 3 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used. 3. Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/ dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Tracking Cell Proliferation Using a Nanotechnology-Based Approach

“…These exogenous reporters are chemical entities which interact with cellular components such as cell membrane (e.g., PKH lipophilic dyes), cytoplasm (e.g., CSFE) and nucleus (e.g., Hoechst 33342), and are distributed to daughter cells after each cell division, in a theoretically equal distribution, resulting in a progressive halving of the progeny fluorescence. This fluorescence intensity reduction can be quantified by conventional fluorescent techniques, the most used one being flow cytometry [5,6]. Although these fluorescent reporters are one of the most common used by the scientific community for the measurement of cell proliferation, they present some limitations such as alteration of the normal function of tracked cells, uneven distribution within the progeny population, rapid dilution during cell proliferation and high cytotoxic effects [5,6].…”

“…This fluorescence intensity reduction can be quantified by conventional fluorescent techniques, the most used one being flow cytometry [5,6]. Although these fluorescent reporters are one of the most common used by the scientific community for the measurement of cell proliferation, they present some limitations such as alteration of the normal function of tracked cells, uneven distribution within the progeny population, rapid dilution during cell proliferation and high cytotoxic effects [5,6]. Consequently, there is a need for a safe and efficient alternative method to track successfully cell proliferation to overcome these limitations.…”

Cell-based in vitro models in environmental toxicology: a review

“…This approach is often more accurate than simple cell counts, as cell density is often variable and reciprocal to proliferative activity in cell culture [24]. A common dye used for this assay is carboxyfluorescein diacetate (CFDA), which has been reported to provide a better resolution than alternative membrane markers like PKH26 [25,26]. Profiles obtained by these assays typically show multiple generation-dependent peaks, defined by fluorescence and cell numbers.…”

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

“…Such CFSE pre-labeling enables the immediate direct comparison of two cell lines or experimental conditions (CFSE-labeled vs. unlabeled) within a single sample tube, reducing variance or subtle differences in incubation time and saving antibody. CFSE is an established fluorescent dye that is commonly used for cell tracking 40 , in proliferation 41,42 and barcoding experiments 43,44 . Finally, while actual sorting steps (FACS, immunomagnetic cell separation or immunopanning) are not part of this protocol, in principle, the harvesting and labeling procedures described here do yield samples that can be subjected to surface antigen-or intracellular labeling-based sorting applications 15 25,28 .…”