Authors: Kimi Honma & Takahiro Ochiya
To date, multiplexing cell-based assay is essential for high-throughput screening of molecular targets. Measuring multiple parameters of a single sample increases consistency and decrease time and cost of assay. Functional assay of living cell is useful as a first step of multiplexing assay, because live-cell assay allows following second assay using cell lysate or stained cell. However, live-cell assay of drug resistant cells that are highly activated of drug efflux mechanisms is sometimes unstable or difficult; for example, the method of measuring colored formazan products by living cells does not show correlation between the amount of formazan and the cells numbers. To this end, more reliable method to allow live-cell assay is anticipated. We described here the protocol of live-cell luciferase assay as a first step of multiplexing cell-based assay of drug-resistant cells which expresses firefly luciferase. This method has several advantages; 1) In growth assay of P-glycoprotein-overexpressing multidrug-resistant cells, firefly luciferase bioluminescence from live cells correlates with the cells numbers; 2) Live-cell assay allows performing second assays, such as a caspase assay, Hoechst staining, and a cell-direct real-time RT-PCR, using the same cells. If necessary, it is possible to continue the cell culture replacing the assay solution with fresh medium; 3) Live-cell luciferase assay is available for other mammalian cells besides drug-resistant cells.
Cells: We used bioluminescent MCF7-ADR-Luc cells that are P-glycoprotein-overexpressing multidrug-resistant beast cancer cells and stably transfected with firefly luciferase gene. MCF7-ADR-Luc cells were cultured in an RPMI 1640 (Gibco BRL) supplemented with 10% fetal bovine serum (Gibco BRL). Luciferin solution: 1 mM beetl luciferin, potassium salt (Promega, E1602) in cell culture medium. Prepare just before assay and equilibrate to room temperature.
The reagent preparation and measuring procedure will require 1 hour.
The assay yields a linear correlation between cell number and luminescence: up to 50,000 cells per well for 96-well plates within a measurement window of zero to 15 minutes. Activity of luciferase enzymes diminishes under a wide range of assay conditions, such as variable temperature, varied ionic strength, and prolonged exposure in solution.
Low luminescent signal
Low correlation between the luciferase luminescence and the cells numbers
The firefly luciferase luminescence from live cells will be directly proportionally to the number of cells (Figure 1).
We thank RU. Takahashi for his excellent technical work.
Various numbers of MCF7-ADR-Luc cells were seeded in a 96-well plate, after cell adhesion to the bottom of well, firefly luciferase bioluminescence was measured. Values are means ± s.d. n = 6. The firefly luciferase luminescence from live cells showed directly proportionally to the number of cells (r2 = 0.9997).
RPN2 gene confers docetaxel resistance in breast cancer, Kimi Honma, Kyoko Iwao-Koizumi, Fumitaka Takeshita, Yusuke Yamamoto, Teruhiko Yoshida, Kazuto Nishio, Shunji Nagahara, Kikuya Kato, and Takahiro Ochiya, Nature Medicine 14 (9) 939 - 948 17/08/2008 doi:10.1038/nm.1858
Kimi Honma & Takahiro Ochiya, Section for Studies on Metastasis, Japanese National Cancer Center Research Institute
Source: Protocol Exchange (2008) doi:10.1038/nprot.2008.169. Originally published online 4 September 2008.