BioTek July 2019 - 25

The cellular confluence can be seen in Figure 5,
where there is little increase in the fluorescence observed between hour 96 and hour 120.

experiments. The reader is unique in that it has the
capability of both the absorbance measurements
using a dedicated UV-Vis monochromator and microscopic imaging using a 6-position objective turret and LED light cubes. The rapid speed of the absorbance reading adds only seconds to a full 96-well
imaging step, but can provide effective information
regarding cell culture status. Gen5™ Microplate
Reader and Imager Software, besides controlling
reader function, can be used to calculate pH from
previously established pH calibration curves. This
unique combination allows continual real-time monitoring of long-term live cell culture experiments.

Figure 5. Montage images of HCT116-GFP cells in culture over 5 days.
HCT116-GFP cells were seeded at 2000 cells per well into a 96-well plate and
allowed to attach for 24 hours. Montage-images (2 x 2) were made with a 4x
objective every 24 hours using a GFP LED cube.

References

Discussion
These data demonstrate that the Cytation™ 5 Cell
Imaging Multi-Mode Reader is capable of monitoring
cell culture pH in live cell experiments while concurrently imaging. Cell cultures normally become acidic
due to an increase in cell numbers and cellular respiration, resulting in a yellowing in color of media formulations containing phenol red. While the change
in pH for short-term experiments is often negligible,
with long-term live cell experiments increasing cell
numbers and the longer duration can overwhelm
the buffering capacity of the media formulation. The
ability to monitor changes in culture pH this in real
time can allow the researcher to have confidence in
the observed experimental results or abort experiments that have deleterious pH conditions.

Clayton, J (2017) Kinetic Proliferation Assay Using LabelFree Cell Counting, BioTek Instruments Application note.
https://www.biotek.com/resources/ application-notes/
kinetic-proliferation-assay-using-labelfree- cell-counting/

Rhoades, Rodney A.; Bell, David R. (2012). Medical
physiology principles for clinical medicine (4th ed.,
International ed.). Philadelphia, Pa.: Lippincott Williams
& Wilkins. ISBN 9781451110395.

Berthois, Y.; Katzenellenbogen, J. A.; Katzenellenbogen,
B. S. (1986). "Phenol red in tissue culture media is a
weak estrogen: Implications concerning the study of
estrogen-responsive cells in culture" (pdf ). Proceedings
of the National Academy of Sciences of the United
States of America. 83 (8): 2946- 2500. PMC 323325 .
PMID 3458212. doi:10.1073/ pnas.83.8.2496.

Held, P. (2018) Using Phenol Red to assess pH in Tissue
Culture Media. Application note. BioTek Instruments,
www.biotek.com

Monitoring cell numbers or cell confluency over time
is a commonly used to test the efficacy of anti-neoplastic agents in preventing cell growth. The use of
phenol red absorbance to monitor media pH insures
that reported inhibition is the result of the test compound rather than poor tissue culture media status.

About the Author

The Cytation 5 is an ideal platform to monitor phenol red absorbance change with live cell imaging

Paul Held, Ph.D., is Laboratory Manager, Applications
Dept., BioTek Instruments, Inc.

https://www.biotek.com/resources/ application-notes/kinetic-proliferation-assay-using-labelfree-cell-counting/ https://www.biotek.com/resources/ application-notes/kinetic-proliferation-assay-using-labelfree-cell-counting/ http://www.biotek.com

BioTek July 2019

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