Category: Assay Development and Screening
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 actual 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 decrease absorbance at 560 nm while an increase in pH results in an increase in 560 nm absorbance.
In these studies, we use a multimode microplate imager to make both absorbance measurements as well as digital microscopic images of long-term 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 that 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 of eventually results in the loss of acidic CO2 results a dramatic increase in pH.
Data demonstrating the physical characteristics of the dye in different mammalian cell media formulations (DMEM, McCoys, and RPMI) will be provided. In addition to long-term mammalian cell proliferation assays, data showing the result of CO2 gas interruption and bacterial contamination will be provided.
Peter Banks– Scientific Director, BioTek Instruments, Winooski, VT
Peter Banks is currently the Scientific Director at BioTek Instruments, Inc. His responsibilities include the management of the company’s applications team and providing scientific guidance to the senior management team on new technology and emerging trends impacting the microplate market and its peripheral markets of liquid handling and detection. Before joining BioTek in 2008, Banks was employed in a number of roles within PerkinElmer for a decade. These roles included strategic market analysis, management of PerkinElmer’s BioPharma R&D program and Director of R&D for PerkinElmer BioSignal in Montreal, Canada.