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Using Phenol Red to Assess pH in Long-Term Proliferation Assays


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February 05, 2018

Authors: Peter Banks and Paul Held, BioTek Instruments, Inc., Winooski, VT USA


Media formulations for the propagation of in vitro tissue culture often contain the pH indicator dye, phenol red. Phenol red is a dye that exhibits a gradual transition from yellow to red over a pH range of 6.2 to 8.2. Above 8.2, the dye turns a bright fuchsia color. This dye has long been used to quickly assess the status of bulk cultures being propagated for subsequent experimental analysis. However, once an experiment has been initiated, the state of phenol red in the media is typically no longer utilized, particularly with image-based proliferation assays. Here we describe the use of this dye to monitor pH changes in live cell proliferation assays.

Phenol red spectral scans at different pH levels indicate an absorbance peak at 560 nm with significant changes that can be utilized to monitor pH changes in long-term mammalian cell proliferation studies. Measurements at this wavelength obey Beer’s Law and are linear with respect to pathlength. Changes downward in pH result in a decreased absorbance at 560 nm while an increase in pH results in an increased 560 nm absorbance.

In these studies, we use a cell imaging multi-mode reader to make both absorbance measurements as well as digital microscopic images of longterm mammalian cell proliferation experiments. These experiments employ HCT116 cells that express a nuclear H2B-GFP chimera protein which fluorescently tags nuclei, allowing them to be counted using imaged-based analysis. Concurrent absorbance determinations are made using absorbance. In cell cultures seeded at low density, cells can grow and multiply unfettered for as long as 5 days. During this interval, the pH of the media decreased from 7.3 to approximately 7.0. In cultures seeded at higher density, cell growth ceased after 3.5 days and media pH decreased to 6.83.

Monitoring phenol red indicator absorbance can also be used to identify environmental changes. Bacterial contamination results in a rapid decrease in pH as bacteria rapidly consume nutrients. The loss of CO2 gas supply results in a rapid increase in pH with carbon dioxide outgassing. Media formulations are designed to provide a pH 7.4 in conjunction with a defined percentage of augmented CO2 gas. The interruption eventually results in the loss of acidic CO2 causing a dramatic increase in pH.

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