Multiwell stiffness assay for the study of cell responsiveness to cytotoxic drugs
Version of Record online: 6 SEP 2013
© 2013 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 111, Issue 2, pages 396–403, February 2014
How to Cite
Zustiak, S., Nossal, R. and Sackett, D. L. (2014), Multiwell stiffness assay for the study of cell responsiveness to cytotoxic drugs. Biotechnol. Bioeng., 111: 396–403. doi: 10.1002/bit.25097
- Issue online: 19 DEC 2013
- Version of Record online: 6 SEP 2013
- Accepted manuscript online: 23 AUG 2013 07:42AM EST
- Manuscript Accepted: 13 AUG 2013
- Manuscript Revised: 12 AUG 2013
- Manuscript Received: 17 APR 2013
- Eunice Kennedy Shriver National Institute of Child Health and Human Development
- National Institutes of Health
Additional supporting information may be found in the online version of this article at the publisher's web-site.
Figure S1. 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.
Figure S2. 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-hand side) and a photoactivatable nitrophenyl azide (reactive group depicted on the right-hand 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.
Figure S3. 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 collagen-coated 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.
Figure S4. 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.
Figure S5. 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 collagen-coated 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.
Figure S6. 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. Hundred microliters of cell suspension was added to each well; n = 3.
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.