Cell Culture Assays
Introduction
Cell
cultures of various sorts are under wide consideration and development
in
toxicology to replace the use of animals, supplement the use of animals,
and
perhaps
provide information that can be obtained by no other convenient means.
An
Institute of Alternatives to Animal Testing operates at Johns Hopkins
University on funding from a variety of private and public sources to
further
the
application of in vitro methods in toxicology testing and research.
A
variety of in vitro systems based on cell cultures or bacterial cultures
are
available. Perhaps the best known, and the oldest, is the Ames Assay,
developed
by
Bruce Ames to screen chemicals for mutagenic potential. This bacterial
system,
based
on specially designed strains of
S. typhimurium, is
supplemented with rat
microsomes, and the back mutation rate is scored as a function of dose
of
chemical. Because chemicals are identified by mutagenic action, and the
system
uses
mammalian microsomes, the screen is also popularly considered to screen
for carcinogenic potential.
Simple
cell cytotoxicity assays are entering the market. One system is based
on the
release of a cytoplasmic enzyme into the culture medium. The enzyme,
lactate
dehydrogenase, is measured by a simple calorimetric assay. Another test
monitors dead cells by a failure to exclude Trypan blue. These tests are
scored
manually by counting cells on a microscope slide, and are therefore less
popular.
In this experiment, we will examine the effect of heat shock on
the cell’s
ability to resist exposure to cadmium, a known lung toxin. We
will use a simple
test for cell activity (not cell viability) to determine a dose
response curve for
cadmium, in the presence and absence of heat shock. The assay is
the uptake
of a vital stain, Neutral red. Most cells will accumulate NR in
lysosomes. The
take up
NR therefore indicates that the cells have suffered damage. The dye
taken
up by the cells is subsequently extracted and measured. Since the amount
of dye
taken up by the cells is a function of cell number, some indication of
cell
mass is
also necessary to interpret the results. In this experiment, cell mass
will
be determined by a sensitive assay for protein.
Procedure
The
response of the cultures to cadmium chloride solutions will be examined.
Two
plates are set up, so divide yourselves into teams, one team per plate.
The
cultures were exposed prior to the laboratory (about 9 AM today) to a
range of
CdCl
2 concentrations: 0.0, 5, 10, 20, and 40 µM. At the beginning of the
class
period,
the medium will be replaced with medium containing neutral red, to a
final
concentration of 50 µg/mL. After an additional 1-hour exposure, you will
remove
the medium, rinse the cells, and determine the Neutral red uptake.
Follow
the protocol. The amount of NR will be determined spectrophotometrically
Cell Culture
A
culture of rat lung epithelial cells are grown on solid substrate in
sterile
flasks.
The cells are released from the substrate and suspended in medium.
About
50,000 cells are plated per well in 24 well plates. The cells are plated
and
grown at 37°C in a medium of 5% newborn calf serum/defined medium
(F12/DMEM) under 5% CO
2. Typically, the cultures reach
confluence after 24
hrs.
Only cultures at, or near, confluence are used in this experiment.
Heat Shock
Twelve
hours before the experiment, 1 plate of cells will be transferred to a
44°C
incubator for 1 hr. The plate is then returned to the regular 37°C
incubator.
Exposure to Cadmuim Chloride
Replicates of 4 wells were exposed to the following concentrations of
CdCl2
2.
Column 1: Blank, no cells
Column 2: 5 µM Cd
Column 3: 10 µM Cd
Column 4: 20 µM Cd
Column 5: 40 µM Cd
Column 6: Control, no exposure.
Neutral Red Assay
Stock Solutions
- Neutral red (0.5% solution in F12/DMEM medium, prepared for student)
- Destaining solution (1% glacial acetic acid, 49% dH2O and 50% ethanol,
V/V)
- Dulbecco’s phosphate buffered saline (PBS)
Preparation of the NR Medium
- Dissolve NR stock solution in ethanol to final concentration of 5 mg/mL.
Incubate at 37°C for 24 hrs.
- Dilute NR stock solution 1 to 100 in cell medium (F12/DMEM) 2 hrs prior
to application to the medium, and hold at 37°C.
- Filter the NR/medium through a 0.45-u filter to remove filtrate and ensure
sterility.
Assay
- Remove the plates from the incubator (normally, this step would be done
in a
laminar flow safety hood; however, the short period of incubation
required for this step allows us to do this on the bench top). Remove
the
existing medium by aspiration. Replace with 1 mL of the NR medium. Use
a
sterile pipette or sterile pipette tips.
- Return the plates to the incubator for 1 hr at 37°C.
- Observe the plates in the inverted microscope. Describe the cultures by
reference to the control plate.
- Remove medium from cells by gentle aspiration. Add 1.0 mL of PBS to
each well gently. Avoid blasting cells free from the bottom of plate. Rotate
the plates several times over a 5-min period.
- Aspirate the PBS. Repeat with another identical volume of PBS.
- Carefully add 1.0 mL of destaining solution. Gently rotate the plates
several
times
every 5 minutes for 15 min. A plate shaker is available.
- Read the plates on the Bio-Tech microplate reader (instructions attached).
Protein Assay
Carefully aspirate the extraction medium. Add 0.1 mL of 1 N NaOH to each
well.
Swirl the plates to dissolve the protein. Add 1.5 mL Biuret reagent.
Read
in the
plate reader after 15 min. (same settings as the NR assay).
Interpretation of Results
The
absorbance value at 540 nm is the amount of NR taken up by the cells. As
cells
lose viability, they lose the ability to take up NR.
Obtain and attach copies of both 24 well plate reports as tables in your
report.
Plot your values of NR uptake, average of 4 wells versus concentration,
and construct a dose response curve. You may wish to convert the NR uptake
values to a percentage response for purposes of plotting. Plot results of both
plates on the figure. As a separate figure, plot the Biuret absorbance versus
concentration to determine if the toxin caused a lose of cell protein from the
well. Comment specifically on whether heat shock altered the dose response of
the cell to cadmium exposure.
