Multiwell stiffness assay for the study of cell responsiveness to cytotoxic drugs
Silviya Zustiak*, Ralph Nossal, Dan L. Sackett
Program on Physical Biology (PPB), Eunice Kennedy Shriver National Institute of Child Health &
Human Development, National Institutes of Health, 9000 Rockville Pike, Bldg. 9, Bethesda, MD 20892
*Corresponding author: Silviya Zustiak, Tel: 314-977-8331, Fax: 314-977-8288, email:
szustiak@slu.edu, Present address: Biomedical Engineering Department, Parks College of Engineering,
Saint Louis University, 3507 Lindell Blvd, St Louis, MO 63103
Supplemental Materials and Methods
Measuring cell viability with a resazurin assay
For the Resazurin assay, a stock solution of 50 mM resazurin sodium salt (Sigma, St. Louis, MO) in
PBS was further diluted 100 times in PBS and then dispensed at 1:10 Resazurin:medium into each well.
The plates were incubated for up to 2 h in a humidified incubator at 37oC and 5% CO2. Again, 80 μl of
the Resazurin:medium solution was transferred from each well of the stiffness assay to a well of a new
96-well plate. The absorbance at 570 nm – 610 nm was reported as a measure of cell number.
Measuring cell viability with a sulforhodamine B (SRB) assay
For the SRB (Sigma, ST. Louis, MO) assay, the cells were first fixed by adding 50 μl of 25%
trichloroacetic acid per 100 μl of media and incubated for 30 min at 4oC. The plates were washed by
submerging 5 times in water and stained with 100 μl per well of 0.2% SRB in 1% acetic acid for 30 min
at room temperature. Excess stain was removed by submerging the plates 4 times in 1% acetic acid. The
bound SRB was then dissolved in 100 μl of 10 mM unbuffered Tris base for 20 min, and the OD at 565
nm was measured. SRB indiscriminately stains proteins including the collagen coating of the gels. Thus,
we were not able to use the SRB for cells seeded on PA gels.
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Supplemental Figures
a)
b)
2
c)
Supplemental Figure 1: Representative force distance curves acquired with an atomic force microscope
(AFM) for a) 1 kPa PA gel, b) 10 kPa PA gel, and c) 100 kPa PA gel. The filled circles, ●, represent the
approach and open circles, ○, represent withdrawal.
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Supplemental Figure 2: The heterobifunctional crosslinker sulfo-SANPAH is used to covalently attach
collagen to the polyacrylamide gels. Sulfo-SANPAH contains an amine-reactive N-hydroxysuccinimide
(NHS) ester (reactive group depicted on the left side) and a photoactivatable nitrophenyl azide (reactive
group depicted on the right side). To achieve covalent collagen coating, the sulfo-SANPAH is deposited
on top of the polyacrylamide gels and the nitrophenyl azide is activated via UV exposure. The
nitrophenyl azide can then react with the polyacrylamide primarily via insertion into C-H and N-H sites.
In a second step, the primary amines in collagen react with the NHS ester group of the crosslinker to
form stable amide bonds.
a)
b)
Supplemental Figure 3: Phase contrast images, demonstrating collagen-coating efficiency of the gels:
a) cells were not able to adhere and spread on non-coated gels, even though some clusters of rounded
cells were observed at the edge of the gels; and b) cells were able to adhere and spread onto collagencoated gels. The PA gels represented here were 10 kPa and were left non-coated or coated with Collagen
Type I. SY5Y cells were seeded for 24 h prior to imaging. The line bar corresponds to 150 μm.
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10 kPa
100 kPa
100 kPa
ACHN
MCF-7
SY5Y
1 kPa
Supplemental Figure 4: Representative phase contrast images, demonstrating the effect of gel stiffness
on cell spreading: the cells seeded on the 1 kPa gels have a rounded morphology, while cells were able
to spread and extend neuritis, in the case of SY5Y neuroblastoma cells, on gels of 10 and 100 kPa. All
cells were seeded 24 h prior to imaging. The line bar corresponds to 100 μm.
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140
% Viable Cell Number
120
100
80
60
40
20
0
0.001
MTS
Resazurin
Sulforhodamine B
0.01
0.1
1
10
100
Paclitaxel concentration (nM)
Supplemental Figure 5: The kinetic MTS and Resazurin assays are comparable to the established
Sulforhodamine B assay in predicting paclitaxel toxicity and indicated the IC50 for a 96 h exposure to be
~7 nM paclitaxel, which correlates well with literature 1. SRB is a stable end-point assay, but it binds
indiscriminately to protein basic amino acid residues. When used to stain cells on top of the collagencoated PA gels, it stains both the cell proteins and the collagen coating, thus giving erroneous viability
results, making it unsuitable for use in our system. Therefore we elected to perform further assessment
of viable cell number using the MTS assay, even though both kinetic assays performed equally well.
For this experiment, SY5Y cells were seeded on a 96-well unmodified plastic plate for 4 h prior to
paclitaxel administration and then exposed to paclitaxel for 96 h with one change of medium at day 2.
The OD was normalized by that for 0 nM paclitaxel; n = 3.
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OD at 490 nm (Cell metabolic
activity)
a)
4x10^5 cells/ml
1x10^5 cells/ml
1.2
1
2x10^5 cells/ml
0.5x10^5 cells/ml
0.8
0.6
0.4
0.2
0
0
20
40
60
80
100
b)
OD at 490 nm (Cell metabolic
activity)
Paclitaxel concentration (nM)
1x10^5 cells/ml
0.25x10^5 cells/ml
1.2
1
0.5x10^5 cells/ml
0.125x10^5 cells/ml
0.8
0.6
0.4
0.2
0
0
20
40
60
80
100
c)
OD at 490 nm (Cell metabolic
activity)
Paclitaxel concentration (nM)
1x10^5 cells/ml
0.25x10^5 cells/ml
1.2
1
0.5x10^5 cells/ml
0.125x10^5 cells/ml
0.8
0.6
0.4
0.2
0
0
20
40
60
80
100
Paclitaxel concentration (nM)
Supplemental Figure 6: The response of cancer cells to paclitaxel as a function of cell number: a)
SY5Y cells, b) HepG2 cells, and c) MDA-MB-231 cells. All cell types were more responsive to
paclitaxel when seeded at a lower initial density in agreement with the commonly-found reduction in in
vitro drug sensitivity of cancer cells with increase in cell density.2 All cells were seeded on 96-well
plastic plates for 4 h prior to drug administration and then exposed to paclitaxel for 96 h with one
change of media at day 2. Cell viability was measured with an MTS assay. 100 μl cell suspension was
added to each well; n = 3.
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References
1
A. Riccardi, T. Servidei, A. Tornesello, P. Puggioni, S. Mastrangelo, C. Rumi and R. Riccardi,
"Cytotoxicity of paclitaxel and docetaxel in human neuroblastoma cell lines", Eur J Cancer. 31A(4),
494-499 (1995).
2
M. T. Dimanche-Boitrel, H. Pelletier, P. Genne, J. M. Petit, C. Le Grimellec, P. Canal, C. Ardiet,
G. Bastian and B. Chauffert, "Confluence-dependent resistance in human colon cancer cells: Role of
reduced drug accumulation and low intrinsic chemosensitivity of resting cells", Int J Cancer. 50(5), 677682 (1992).
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