BioTek July 2019 - 24

images were made using a 4x objective and stitched
into a single file using Gen5 Microplate Reader and
Imager Software (BioTek Instruments). Primary mask
analysis of the captured digital images were used to
determine the number of cells. Nuclei are identified
as fluorescent objects between 5-100 μm in size and
having fluorescence in excess of a threshold of 1500.
Immediately after imaging, the well absorbance at
560 nm was measured using the UV-Vis monochromator module of the Cytation 5.

Figure 2. Change in HCT116 Cell Count and pH over time in nonconfluent
cultures. HCT116 cells were seeded at a density of 500 cells per well. After
24 hours to allow for attachment, image-based and absorbance analysis were
performed on 96-well plate cultures every 2 hours for 5 days and the results
plotted. pH was determined by interpolating data from a previously generated
calibration curve. Data represents the mean and SEM for 96 determinations at
each data point.

Results
Live HCT116 cells expressing GFP in their nuclei are
easily counted using image analysis. The green fluorescence identifies each cell without the need for a nucleic acid binding stain that could affect cell growth.
When HCT116-GFP cells are seeded at low density
and monitored over the course of five days, the 560
absorbance was used to calculate pH in each well by
interpolating a previously determined pH calibration
curve. The pH in these cultures decreased only about
0.15 over the course of 5 days, despite a 50-fold increase in the number of cells counted (Figure 2).

Figure 3. Montage images of HCT116-GFP cells in culture over 5 days.
HCT116-GFP cells were seeded at 500 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.

In these experiments, cells were seeded at a density
such that at the end of five days they have not reached
confluence (Figure 3). In this state, the cells are not contact inhibited and can freely divide. The only limitation
being media nutrients and media pH status.
When HCT116-GFP cells are seeded at higher density, pH change becomes a greater issue and cell
number increase eventually stalls (Figure 4). When
cells are seeded at 2000 cells per well, the increased
number of cells causes the pH to drop about 2-fold
more than the lower seeding density. This drop in pH
continues the length of the experiment despite the
cell number stalling after about 80 hours. At higher
cell densities, cells continue to respire, despite the
cessation of cell number increase, resulting in a decrease in the pH.

Figure 4. Change in HCT116 Cell Count and pH over time in confluent
cultures. HCT116 cells were seeded at a density of 2000 cells per well.
After 24 hours to allow for attachment, image-based and absorbance
analysis performed on 96-well plate cultures every 2 hours for 5 days. pH
was determined by interpolating the 415/560 ratio values with a previously
generated calibration curve. Data represents the mean and SEM for 96
determinations at each data point.

BioTek July 2019

Table of Contents for the Digital Edition of BioTek July 2019