A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin
Author(s)Kulkarni, Chethana; Betts, Alison M.; Maass, Katie F.; Quadir, Mohiuddin Abdul; Hammond, Paula T.; Wittrup, Karl Dane; ... Show more Show less
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Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell’s ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 10[superscript 5]–10[superscript 10] doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell’s ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC[subscript 50] values of 4–12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug’s single-cell potency and can be used for any fluorescent or fluorescently labeled drug, including nanoparticles or antibody–drug conjugates.
DepartmentMassachusetts Institute of Technology. Department of Biological Engineering; Massachusetts Institute of Technology. Department of Chemical Engineering; Koch Institute for Integrative Cancer Research at MIT
Journal of Pharmaceutical Sciences
Maass, Katie F. et al. “A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin.” Journal of Pharmaceutical Sciences 104.12 (2015): 4409–4416.
Author's final manuscript