Name: Comprehensive Analysis of Drug Response using the FLICK Assay (Video) | JoVE Journal
Uploaded: 2025-06-06T13:00:00.000+00:00
Duration: 9 min 42 s
Description: Learn the FLICK assay for comprehensive drug response analysis in cancer research. This method calculates precise growth and death rates to distinguish live from dead cells.

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5 minutes

We thank all past and present members of the Lee Lab for their contributions to our lab&#39;s perspective on evaluating drug responses. This work was supported by funding from the National Institutes of Health to MJL (R21CA294000 and R35GM152194).

<ol><li>Hafner, M., Niepel, M., Sorger, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alternative+drug+sensitivity+metrics+improve+preclinical+cancer+pharmacogenomics%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Alternative drug sensitivity metrics improve preclinical cancer pharmacogenomics.</a> Nat Biotechnol. 35 (6), 500-502 (2017).</li>
<li>Dixon, S. J., Lee, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quick+tips+for+interpreting+cell+death+experiments%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Quick tips for interpreting cell death experiments.</a> Nat Cell Biol. 25 (12), 1720-1723 (2023).</li>
<li>Schwartz, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drug+GRADE%3A+An+Integrated+Analysis+of+Population+Growth+and+Cell+Death+Reveals+Drug-Specific+and+Cancer+Subtype-Specific+Response+Profiles%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Drug GRADE: An Integrated Analysis of Population Growth and Cell Death Reveals Drug-Specific and Cancer Subtype-Specific Response Profiles.</a> Cell Rep. 31 (12), 107800(2020).</li>
<li>Hafner, M., Niepel, M., Chung, M., Sorger, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growth+rate+inhibition+metrics+correct+for+confounders+in+measuring+sensitivity+to+cancer+drugs%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Growth rate inhibition metrics correct for confounders in measuring sensitivity to cancer drugs.</a> Nat Methods. 13 (6), 1-11 (2016).</li>
<li>Galluzzi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+mechanisms+of+cell+death+recommendations+of+the+Nomenclature+Committee+on+Cell+Death+2018%5BTitle%5D)+AND+%22Cell+Death+Differ%22%5BJournal%5D)">Molecular mechanisms of cell death recommendations of the Nomenclature Committee on Cell Death 2018.</a> Cell Death Differ. 25 (3), 486-541 (2018).</li>
<li>Tsvetkov, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Copper+induces+cell+death+by+targeting+lipoylated+TCA+cycle+proteins%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Copper induces cell death by targeting lipoylated TCA cycle proteins.</a> Science. 375 (6586), 1254-1261 (2022).</li>
<li>Holze, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxeiptosis%2C+a+ROS-induced+caspase-independent+apoptosis-like+cell-death+pathway%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">Oxeiptosis, a ROS-induced caspase-independent apoptosis-like cell-death pathway.</a> Nat Immunol. 19 (2), 130-140 (2018).</li>
<li>Maltese, W. A., Overmeyer, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methuosis+Nonapoptotic+Cell+Death+Associated+with+Vacuolization+of+Macropinosome+and+Endosome+Compartments%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">Methuosis Nonapoptotic Cell Death Associated with Vacuolization of Macropinosome and Endosome Compartments.</a> Am J Pathol. 184 (6), 1630-1642 (2014).</li>
<li>Inde, Z., Forcina, G. C., Denton, K., Dixon, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetic+Heterogeneity+of+Cancer+Cell+Fractional+Killing%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Kinetic Heterogeneity of Cancer Cell Fractional Killing.</a> Cell Rep. 32 (1), 107845(2020).</li>
<li>Forcina, G. C., Conlon, M., Wells, A., Cao, J. Y., Dixon, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systematic+Quantification+of+Population+Cell+Death+Kinetics+in+Mammalian+Cells%5BTitle%5D)+AND+%22Cell+Syst%22%5BJournal%5D)">Systematic Quantification of Population Cell Death Kinetics in Mammalian Cells.</a> Cell Syst. 4 (6), 1-18 (2017).</li>
<li>Gelles, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-Cell+and+Population-Level+Analyses+Using+Real-Time+Kinetic+Labeling+Couples+Proliferation+and+Cell+Death+Mechanisms%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Single-Cell and Population-Level Analyses Using Real-Time Kinetic Labeling Couples Proliferation and Cell Death Mechanisms.</a> Dev Cell. 51 (2), 277-291.e4 (2019).</li>
<li>Richards, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drug+antagonism+and+single-agent+dominance+result+from+differences+in+death+kinetics%5BTitle%5D)+AND+%22Nat+Chem+Biol%22%5BJournal%5D)">Drug antagonism and single-agent dominance result from differences in death kinetics.</a> Nat Chem Biol. 16 (7), 791-800 (2020).</li>
<li>Richards, R., Honeywell, M. E., Lee, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FLICK%3A+an+optimized+plate+reader-based+assay+to+infer+cell+death+kinetics%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">FLICK: an optimized plate reader-based assay to infer cell death kinetics.</a> STAR Protoc. 2 (1), 100327(2021).</li>
<li>Honeywell, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+genomic+screens+with+death+rate+analyses+reveal+mechanisms+of+drug+action%5BTitle%5D)+AND+%22Nat+Chem+Biol%22%5BJournal%5D)">Functional genomic screens with death rate analyses reveal mechanisms of drug action.</a> Nat Chem Biol. 20 (11), 1443-1452 (2024).</li>
<li>Jannoo, R., Xia, Z., Row, P. E., Kanamarlapudi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+of+the+Interleukin-13+Receptor+%28IL-13R%29%CE%B12+Expressing+Prostate+Cancer+by+a+Novel+Hybrid+Lytic+Peptide%5BTitle%5D)+AND+%22Biomolecules%22%5BJournal%5D)">Targeting of the Interleukin-13 Receptor (IL-13R)α2 Expressing Prostate Cancer by a Novel Hybrid Lytic Peptide.</a> Biomolecules. 13 (2), 356(2023).</li>
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</ol>

The authors have no conflicts of interest to disclose.

The FLICK assay is a robust method for generating a comprehensive evaluation of a drug&#39;s effect on the growth and death of a cell population. Because this method does not directly count cells, critical steps in the protocol are to ensure assay linearity and complete lysis during the triton permeabilization steps. The correct permeabilization time can be identified visually, as highlighted in this protocol, or quantitatively by reading plate fluorescence over time and identifying when the signal plateaus. In our exper...

For anti-cancer drugs, pre-clinical evaluation of drug sensitivity generally involves testing how drugs affect the viability of cells in culture1. Cell viability following drug exposure is a product of at least two separate effects: drug-induced inhibition of cell proliferation and activation of cell death2. Unfortunately, although cell death is a critical feature that is required for durable drug responses, standard approaches fail to clarify the degree to which a drug activates cell death3.
Common drug response assays include those that directly count cells (e.g., Coulter Counter, some uses of flow cytometry or microscopy), quantify the ability of cells to proliferate (e.g., colony formation assay), or quantify a metabolic activity (e.g., CellTiter-Glo, tetrazolium based MTT or MTS assays). A shared feature of these assays is that the data generated is proportional to the number of live cells. Because drugs vary considerably in how they coordinate growth inhibition and cell death, the number of live cells following drug exposure provides an unreliable insight into the level of drug-induced cell death3. Furthermore, because cancer cells generally proliferate rapidly in cell culture, the number of live cells can be dramatically reduced relative to the untreated population without inducing any cell death4. Thus, a central flaw is that the degree of cell death cannot be quantified without measuring both the number of live and dead cells.
Quantifying the number of dead cells following drug exposure is challenging to do accurately for several reasons2. First, more than a dozen regulated cell death pathways exist5,6,7,8. Although biochemical markers generally exist for identifying each type of regulated cell death, these markers vary in their specificity, and no single assay can be used to simultaneously quantify all death subtypes. Secondly, the timing of activation for each form of cell death can vary quite dramatically depending on the context, so a complete picture cannot emerge unless death is quantified over time9,10. Many biochemical assays for quantifying cell death produce an end-point measurement, so generating kinetic data can be challenging and limited by cost. A third complication is that the dead cell itself is a transient intermediate state between the live cell state and dissociated cell debris. The stability of dead cells varies depending on the death subtype, with some types, such as apoptosis, creating relatively stable corpses, whereas other types of death cause rapid lysis. Thus, methods of quantifying death that require collection and counting of dead cells also will produce a biased understanding of cell death. Finally, a fourth limitation is that biochemical assays that quantify the degree of cell death typically fail to provide any insight into how a drug alters proliferation. Thus, the overall population size - and, importantly, whether the population is expanding or shrinking - cannot be interpreted.
Some microscopy-based assays, such as STACK and SPARKL, are effective at measuring live and dead cells over time, and these assays can produce comprehensive insights about drug-induced cell death10,11. These assays, however, require specialized instruments, such as the Incucyte microscope, creating limitations in throughput and access to these approaches. Additionally, microscopy-based techniques require that dead cells remain in the focal plane of the microscope throughout the duration of the experiment, compromising the ability to quantify dead cells when they lose adherence from the plate or over time as dead cells decay. Similarly, microscopy-based assays face challenges when applied in the context of suspension cultures, as cells drift in and out of a given focal plane.
To address the issues highlighted above, we have generated an assay called FLICK (Fluorescence-based and Lysis-dependent Inference of Cell Death Kinetics)12,13. The goal of the FLICK assay is to determine the level of drug-induced cell death, regardless of how the cells are dying. The FLICK method uses cell impermeant dyes, whose fluorescence depends on DNA binding. A key feature of FLICK is the use of these fluorophores to label dead cell accumulation over time by virtue of their accessible DNA, followed by a mechanical detergent-based lysis to permeabilize any live cells at the end of the assay. These data, combined with mathematical modeling, enable the quantification of both live and dead cell populations with continuous temporal resolution and without requiring the collection or handling of dead cells. Furthermore, the use of a plate reader to evaluate dead cell fluorescence allows for the evaluation of dead cells without requiring that dead cells remain intact, thus alleviating a bias against necrotic forms of death that result in cell rupture. Finally, the FLICK assay requires minimal plate handling and can rapidly generate kinetic measurements, allowing for high-throughput drug screening. In this protocol, we focus on the use of the FLICK assay, including how to use FLICK to infer the drug-induced growth rate, death rate, and/or the mechanisms of cell death.

<table><tbody><tr><td>Belinostat</td><td>ApexBio</td><td>A4612</td><td></td></tr><tr><td>Camptothecin</td><td>ApexBio</td><td>A2877</td><td></td></tr><tr><td>Conical centrifuge tube, 15mL</td><td>Fisher Scientific</td><td>12-565-269</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10017CV</td><td>For seeding and drugging cells</td></tr><tr><td>DMSO</td><td>Fisher Scientific</td><td>MT-25950CQC</td><td>For seeding and drugging cells</td></tr><tr><td>Fisherbrand 96-Well, Cell Culture-Treated, U-Shaped-Bottom Microplate</td><td>Fisher Scientific</td><td>FB012932</td><td>For seeding and drugging cells (pin plate)</td></tr><tr><td>Greiner Bio-One&amp;nbsp;CELLSTAR &amp;mu;Clear 96-well, Cell Culture-Treated, Flat-Bottom Microplate</td><td>Greiner</td><td>655090</td><td>For seeding and drugging cells</td></tr><tr><td>IncuCyte S3</td><td>Essen Biosciences</td><td></td><td>Any phase microscope will work</td></tr><tr><td>MATLAB</td><td>MathWorks</td><td>https://www.mathworks.com/products/matlab.html</td><td>MATLAB version R2023b, a license is required</td></tr><tr><td>Microplate fluorescence reader</td><td>Tecan</td><td>Spark</td><td>For measuring dead cell fluorescence</td></tr><tr><td>Palbociclib</td><td>ApexBio</td><td>A8316</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CM</td><td>Any PBS works</td></tr><tr><td>Spark Multimode Microplate Reader</td><td>Tecan</td><td>https://www.tecan.com/spark-overview</td><td>SparkControl software version 2.2</td></tr><tr><td>Sterile reservoir, 25 mL</td><td>Fisher Scientific</td><td>13-681-508</td><td>For seeding and drugging cells</td></tr><tr><td>Sytox Green nucleic acid stain</td><td>Thermo Fisher Scientific</td><td>S7020</td><td>DNA stain for measuring dead cell fluorescence</td></tr><tr><td>Triton-X 100</td><td>Thermo Fisher Scientific</td><td>J66624-AP</td><td>For permeabilizing cells</td></tr><tr><td>U2OS</td><td>ATCC</td><td>HTB-96</td><td>An example cell line used in this protocol</td></tr><tr><td>Z-VAD-FMK</td><td>ApexBio</td><td>A1902</td><td></td></tr></tbody></table>

comprehensive analysis of drug response using the flick assay

Comprehensive Analysis of Drug Response using the FLICK Assay

For understanding drug efficacy, a critical need is to characterize the extent of drug-induced cell death. Efforts to quantify the level of drug-induced cell death are challenged by the existence of more than a dozen molecularly distinct forms of regulated death, each with its own activation timing and biochemical hallmark features. Furthermore, for some necrotic death subtypes, hallmark features are only observed transiently and are rapidly lost due to cell rupture. Thus, even when using a combination of death pathway-specific assays, it is challenging to accurately quantify the total amount of cell death or the relative contributions of each death subtype. Another issue is that many death-specific assays ignore how drugs affect cell proliferation, making it challenging to interpret if a drug-treated population is expanding or shrinking. The FLICK assay allows for quantification of the total level of cell death following stimulation in a manner that is specific to death but also largely agnostic to the type(s) of death activated. Additionally, the FLICK assay retains information about the total population size and cell proliferation rate. In this manuscript, we describe the basic use of the FLICK assay, how to troubleshoot this assay when using different types of biological material, and how to use the FLICK assay to quantify the contributions of each type of cell death to an observed drug response.

Using this protocol, we explored the sensitivity of U2OS cells to the HDAC inhibitor Belinostat. These experiments were performed using 2 µM SYTOX Green to label dead cells (Figure 1A). Kinetic readings were made using a fluorescent plate reader at a 130 gain setting (Figure 1B). Cells were lysed in 1.5% Triton-X solution in PBS for 2 h at the end of the assay (Figure 1C-D).
The FLICK pro...

This protocol describes how to use the FLICK assay for evaluating drug responses, including detailed instructions for using this assay to compute the drug-induced growth rates and death rates and to evaluate the mechanism of drug-induced cell death.

For understanding drug efficacy, a critical need is to characterize the extent of drug-induced cell death. Efforts to quantify the level of drug-induced ...

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Learn the FLICK assay for comprehensive drug response analysis in cancer research. This method calculates precise growth and death rates to distinguish live from dead cells.

Comprehensive Analysis of Drug Response using the FLICK Assay

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