BioTek July 2019 - 14

Technologies (Rockville, MD).
Sterile 96-well clear-bottom microplates, catalog number 3603,
were obtained from Corning
(Corning, NY). Culture media, fetal bovine serum, insulin, and
antibiotics were purchased from
Invitrogen (Carlsbad, CA).
Mouse C-10 lung epithelial cells
were cultured in DMEM/F12
supplemented with 10% fetal
bovine serum (FBS). Cells were
trypsinized and re-suspended in
fresh media at a concentration of
75,000 cells/mL. Serial dilutions
(1:2) were made using DMEM/
F12 with 10% FBS as the diluent.
Aliquots (200 µL) of each cell dilutions were then pipetted into
96-well microplates. Cells were
allowed to grow overnight at
37°C in a humidified 5% CO2 environment. The following day the
cells were quantitated using a cell
counting kit CCK-8. Cell quantitation was performed according to
the assay kit instructions. Briefly,
20 µL of the ready to use kit reagent is added directly to cell cultures in 96-well plates that have
200 µL of media. Cells are incubates in a humidified environment at 37°C, 5% CO2 for 2 hours.
After incubation, the absorbance
at 460 nm was determined using
a Synergy Multi-Mode Microplate
Reader (BioTek Instruments).
For cytotoxic agent studies,

Primary human mesothelioma
cells were seeded at 5,000 cells
per well in a 200 µL volume in
DMEM/F12 media supplemented with 10% FBS. After allowing
cells to grow for 48 hours at 37°C
in a humidified 5% CO2 environment, various concentrations (0
to 40 µM) of the antibiotic, thiostrepton were added to the cells.
Following a 24 hour exposure to
the drug, 10 µL of the cell counting reagent was added directly
to the cell cultures. The plates
were incubated for 120 minutes
at 37°C in a humidified 5% CO2
environment. The WST-8 formazan product was measured at
460 nm using a Synergy MultiMode Microplate Reader as described previously.

Spectral scans of reacted and
unreacted cell cultures were
performed using a Synergy
Multi-Mode Microplate Reader.
Reacted wells contained approximately 20,000 primary mesothelioma cells while unreacted wells
only had media. Absorbance
measurements were made from
300 nm to 700 nm in 1 nm increments. In all experiments, the
reader was controlled by and
the data collected using Gen5™
Data Analysis Software (BioTek
Instruments, Winooski, VT).

Results
An absorbance spectral scan of
reacted WST-8 formazan product, as well as unreacted WST-8 in

Figure 2. Spectral Scan of WST-8 Formazan. The absorbance spectra from 300 nm to 700 nm in 1-nm
increments was measured in reacted samples with WST-8 formazan product (magenta) or non reacted
WST-8 (blue).

BioTek July 2019

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