Quantitative analysis of cell proliferation by a dye dilution assay: Application to cell lines and cocultures

Abstract

Cell proliferation represents one of the most fundamental processes in biological systems, thus the quantitative analysis of cell proliferation is important in many biological applications such as drug screening, production of biologics, and assessment of cytotoxicity. Conventional proliferation assays mainly quantify cell number based on a calibration curve of a homogeneous cell population, and therefore are not applicable for the analysis of cocultured cells. Moreover, these assays measure cell proliferation indirectly, based on cellular metabolic activity or DNA content. To overcome these shortcomings, a dye dilution assay employing fluorescent cell tracking dyes that are retained within cells was applied and was diluted proportionally by subsequent cell divisions. Here, it was demonstrated that this assay could be implemented to quantitatively analyze the cell proliferation of different types of cell lines, and to concurrently analyze the proliferation of two types of cell lines in coculture by utilizing cell tracking dyes with different spectral characteristics. The mean division time estimated by the dye dilution assay is compared with the population doubling time obtained from conventional methods and values from literature. Additionally, dye transfer between cocultured cells was investigated and it was found that it is a characteristic of the cells rather than a characteristic of the dye. It was suggested that this method can be easily combined with other flow cytometric analyses of cellular properties, providing valuable information on cell status under diverse conditions. © 2017 International Society for Advancement of Cytometry.

Tracking Cell Proliferation Using a Nanotechnology-Based Approach

“…The main advantages of using nanoparticles rather than classical approaches are that: a) they are more stable than fluorophore in solution [15], b) the fluorescence signal does not decreases rapidly during the first 24 hours after labelling, then zeroth generation can be set up using cell sample from day 0 [23] and c) they can be used to track a broad range of cellular types without any cytotoxic effect allowing its application in many biological/biomedical research fields [18]. To the best of our knowledge, cell clone formation assay based on nanotechnology imaging technique by using Quantum dot, has been the only reported to date to study the proliferative features [19].…”

Characteristics of live parameters of the HS‐5 human bone marrow stromal cell line cocultured with the leukemia cells in hypoxia, for the studies of leukemia–stroma cross‐talk

“…For the 72‐h co‐culture experiments, we strived to perform the studies in as physiological conditions as possible, so we utilized the viable cell trackers instead of the stable expression of proteins such as GFP or RFP. To do this, we chose staining combinations that allowed a safe, nontoxic, and stable cell tracking with the use of dyes recommended for the quantitative analysis of proliferation as well as for the long‐term cell tracking experiments . As those dyes bind to such intracellular components such as proteins and lipids, their staining efficiency does not depend on any specific antigens or markers.…”

“…To visualize and track HS‐5 and K562 cells in co‐culture, HS‐5 cells were seeded on a collagen precoated glass bottom dish (MatTek Corporation) and labeled with 15 μM CMAC (Thermo Fisher Scientific, Poland) or 10 μM cell proliferation dye eFluor670 or eFluor450 (eBioscience, Invitrogen/Thermo Fisher Scientific, Poland) or CFSE (Thermo Fisher Scientific), according to the manufacturer's protocols. According to the provided product information, all these probes can be used for the long‐term cell tracking . After labeling and washing, the cells were left in the culture media in incubator for at least 2 h, followed by the media exchange and start of co‐culture with K562 cells.…”

“…To assess proliferation, a dye dilution assay was utilized . Cells were labeled with 10 μM cell proliferation dye eFluor670, eFluor450, or CFSE, according to the manufacturer's protocol.…”

Alexidine dihydrochloride has broad spectrum a ctivities against diverse fungal pathogens

“…Cells were stained with 2 mM CFSE (Carboxyfluorescein Succinimidyl ester) for 5 minutes and washed with 1X RPMI media three times. This dye is commonly used to measure cell proliferation; with each cell division the amount of CFSE is diluted in half, which can be observed via flow cytometry (37). After the staining, the cells were counted and adjusted at cell density of 5X10 6 cells/ml and plated 100 μl/well in a round bottom 96-well plate.…”