BioTek July 2019 - 18

Cell Imaging
Cell culture plates were transferred by the BioSpa
8 to a Cytation™ 5 Cell Imaging Multi-Mode Reader
(BioTek Instruments, Winooski, VT) every two hours.
Environmental conditions were maintained at 37 ºC
and 5% CO2 within the Cytation 5 throughout the
imaging steps. Two high contrast brightfield images
were captured at each time point: an in focus image
used for reference, and a defocused image for cell
counting3. Briefly, cells were brought into focus using the High Contrast Brightfield kit and the "in focus" focal height recorded. The View Line Profile tool
was then used to draw a line that crossed cells and
background sections of the imaging field without
cells present. The focal height was then decreased
while observing the line profile to determine the focal height at which maximum contrast between cell
and background brightness was achieved.

Figure 2. Comparison of high contrast brightfield label-free cell counting to
cell counts using Hoechst labeled nuclei. Cell counts of Hoechst stained
NIH3T3 cells from the two techniques were highly comparable across a
wide range of seeding densities. Points were fitted to a nonlinear regression
equation with an R2 value of 0.99 and a slope of 0.97.

NIH3T3, HCT116, and HeLa cell growth was monitored for five days. All three cell types exhibited
robust logarithmic growth up to full confluence.
To demonstrate the ability of this system to screen
pharmacological agents, cell proliferation profiles
for cells cultured with eight concentrations of two
anti-cancer drugs were generated. Concentrationresponse curves and IC50 values were used to quantify anti-proliferation effects for each cell type.

Image Analysis
Image preprocessing was used to obtain the best
possible enhancement of contrast, reducing each
cell to a single bright spot. Object masking thresholds were then set to identify each cell for counting
(Table 1).

Materials and Methods
Cell Culture
NIH3T3 and HeLa cells were grown in Advanced
Dulbecco's Modified Eagle's Medium (DMEM)
(Gibco, Grand Island, NY) with 10% FBS (Gibco),
and 1x PenStrep-Glutamine (Cellgro, Manassas, VA).
HCT116 cells were grown in McCoys 5A medium
(Gibco) with 10% FBS, and 1x PenStrep-Glutamine.
Cells were seeded into black sided, clear bottom
96-well microplates (Corning, Corning, NY) at 2000
cells per well. Environmental conditions, including
temperature (37 °C), gas (5% CO2) and humidity
(90%) were maintained during the five day incubation by a BioSpa™ 8 Automated Incubator (BioTek
Instruments, Winooski, VT).

Results
Quantitative Evaluation of Cell Proliferation
Using Label-Free Direct Cell Counting
Label-free methods of measuring cell growth kinetics are preferable over the use of stains that can influence proliferation rates. Although confluence level can be used for some applications, cell counting is
the most direct quantitative measure of cell proliferation over a broad range of cell population densities
(Figure 3).

BioTek July 2019

Table of Contents for the Digital Edition of BioTek July 2019