| Autor: |
Trumpi K; Department of Surgery,, University Medical Center Utrecht, Heidelberglaan 100, 3584CX Utrecht, The Netherlands., Egan DA; Cell Screening Core, Department of Cell Biology, Centre for Molecular Medicine, University Medical Center Utrecht, Heidelberglaan 100, 3584CX Utrecht, The Netherlands., Vellinga TT; Department of Surgery,, University Medical Center Utrecht, Heidelberglaan 100, 3584CX Utrecht, The Netherlands., Borel Rinkes IH; Department of Surgery,, University Medical Center Utrecht, Heidelberglaan 100, 3584CX Utrecht, The Netherlands., Kranenburg O; Department of Surgery,, University Medical Center Utrecht, Heidelberglaan 100, 3584CX Utrecht, The Netherlands. |
| Abstrakt: |
Novel spheroid-type tumor cell cultures directly isolated from patients' tumors preserve tumor characteristics better than traditionally grown cell lines. However, such cultures are not generally used for high-throughput toxicity drug screens. In addition, the assays that are commonly used to assess drug-induced toxicity in such screens usually measure a proxy for cell viability such as mitochondrial activity or ATP-content per culture well, rather than actual cell death. This generates considerable assay-dependent differences in the measured toxicity values. To address this problem we developed a robust method that documents drug-induced toxicity on a per-cell, rather than on a per-well basis. The method involves automated drug dispensing followed by paired image- and FACS-based analysis of cell death and cell cycle changes. We show that the two methods generate toxicity data in 96-well format which are highly concordant. By contrast, the concordance of these methods with frequently used well-based assays was generally poor. The reported method can be implemented on standard automated microscopes and provides a low-cost approach for accurate and reproducible high-throughput toxicity screens in spheroid type cell cultures. Furthermore, the high versatility of both the imaging and FACS platforms allows straightforward adaptation of the high-throughput experimental setup to include fluorescence-based measurement of additional cell biological parameters. |
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