Name: an automated differential nuclear staining assay for accurate determination of mitocan cytotoxicity
Uploaded: 2020-05-12T15:00:00
Description: Watch this Scientific Journal Video about An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity at JoVE.com

NVK, a CPRIT scholar in Cancer Research, thanks the Cancer Prevention and Research Institute of Texas (CPRIT) for their generous support, CPRIT grant RR150044. This work was also supported by the Welch Foundation Research Grant C-1930, and by the National Institutes of Health R35 GM129294 awarded to NVK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1. Cytotoxicity Assay: Setup
<ol>
	<li>Prepare solutions of compounds of interest at desired concentrations in the appropriate media (serum-free or 1, 2.5, or 5% FBS RPMI-1640).
	<ol>
		<li>To measure cytotoxicity of a single compound (e.g., to determine effective doses), prepare compounds at 2x final concentration.</li>
		<li>To measure cytotoxicity of compound combinations, prepare compounds at 4x final concentration.</li>
		<li>Prepare solvent-only controls by mixing the same amount of solvent with the appropriate m...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulators+of+mitochondrial+dynamics+in+cancer.">Regulators of mitochondrial dynamics in cancer.</a> Current Opinion in Cell Biology. 39, 43-52 (2016).</li><li>Vazquez, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PGC1&#945;+expression+defines+a+subset+of+human+melanoma+tumors+with+increased+mitochondrial+capacity+and+resistance+to+oxidative+stress.">PGC1&#945; expression defines a subset of human melanoma tumors with increased mitochondrial capacity and resistance to oxidative stress.</a> Cancer Cell. 23 (3), 287-301 (2013).</li><li>Caino, M. C., Altieri, D. 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J., Rodr&#237;guez-Enr&#237;quez, S., Neuzil, J., Saavedra, E., Moreno-S&#225;nchez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+causes+of+cancer+revisited:+"mitochondrial+malignancy"+and+ROS-induced+oncogenic+transformation+-+why+mitochondria+are+targets+for+cancer+therapy.">The causes of cancer revisited: "mitochondrial malignancy" and ROS-induced oncogenic transformation - why mitochondria are targets for cancer therapy.</a> Molecular Aspects of Medicine. 31 (2), 145-170 (2010).</li><li>Kreitz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Plasticity+of+Acute+Myeloid+Leukemia.">Metabolic Plasticity of Acute Myeloid Leukemia.</a> Cells. 8 (8), (2019).</li><li>Sriskanthadevan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AML+cells+have+low+spare+reserve+capacity+in+their+respiratory+chain+that+renders+them+susceptible+to+oxidative+metabolic+stress.">AML cells have low spare reserve capacity in their respiratory chain that renders them susceptible to oxidative metabolic stress.</a> Blood. 125 (13), 2120-2130 (2015).</li><li>Guerra, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Dysfunction:+A+Novel+Potential+Driver+of+Epithelial-to-Mesenchymal+Transition+in+Cancer.">Mitochondrial Dysfunction: A Novel Potential Driver of Epithelial-to-Mesenchymal Transition in Cancer.</a> Frontiers in Oncology. 7, 295 (2017).</li><li>Guerra, F., Arbini, A. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+therapy:+Targeting+mitochondria+and+other+sub-cellular+organelles.">Cancer therapy: Targeting mitochondria and other sub-cellular organelles.</a> Current Pharmaceutical Design. 20 (2), 201-222 (2014).</li><li>Yamaguchi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+elimination+of+cancer+cells+by+deoxyglucose-ABT-263/737+combination+therapy.">Efficient elimination of cancer cells by deoxyglucose-ABT-263/737 combination therapy.</a> PLoS One. 6 (9), 24102 (2011).</li><li>Hahn, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+anti-cancer+drugs,+mitocans,+to+enhance+the+immune+responses+against+tumors.">Use of anti-cancer drugs, mitocans, to enhance the immune responses against tumors.</a> Current Pharmaceutical Biotechnology. 14 (3), 357-376 (2013).</li><li>Panina, S. B., Pei, J., Baran, N., Konopleva, M., Kirienko, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+Synergistic+Potential+of+Mitochondria-Targeting+Drugs+for+Leukemia+Therapy.">Utilizing Synergistic Potential of Mitochondria-Targeting Drugs for Leukemia Therapy.</a> Frontiers in Oncology. 10, 435 (2020).</li><li>Lema, C., Varela-Ramirez, A., Aguilera, R. 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Although the protocol for Hoechst/PI cytotoxicity assay is robust and requires comparatively little hands-on time, there are several experimental details that are very important to ensure accurate results. First, it is essential to make sure that the concentration of DMSO remains below 0.5% (v/v). It is generally agreed that exposure to even low doses of DMSO can substantially alter the morphology and attachment of cells and significantly delay cell cycle progression39,<sup class="xref"...

The first step to identifying effective cancer treatments is the selection of a robust, unbiased cytotoxicity assay that can be used to examine the effect of treatment. A common choice for low-throughput experiments is the exclusion of trypan blue dye from living cells. This method is favored because it allows a relatively unbiased method for quantifying cell survival. trypan blue passively diffuses into cells whose membranes are compromised, but it is effectively blocked from entering healthy cells1. The quotient of the living cells and the total cells represents the percent viability, which indicates the efficacy of the treatment. The most significant disadvantage of the trypan blue assay is that it is poorly suited for high-throughput methodologies. It has a relatively low signal-to-noise ratio and prolonged staining can result in artifacts due to the staining of viable cells. Consequently, trypan blue exclusion is typically, but not always2, relegated to manual counting. This makes it too slow and introduces the&#160;strong possibility of bias due to subjective judgment of the researcher (unless blinding or independent counts are used, which further reduce laboratory throughput). In general, the throughput of this assay is insufficient for modern drug discovery.
Viability assays, which generally have a much higher throughput, allow researchers to circumvent this limitation, but come with significant caveats (see&#160;Table 1). These methods generally fall into two groups. One group is comprised of colorimetric assays that are based on the function of cellular redox enzymes. Colorless or non-fluorescent substrates are converted into vibrant products that can be quantified using a spectrophotometer. Classic examples include tetrazolium salts (MTT, WST-1, XTT, etc.) and resazurin. This category also includes luminescent assays that utilize luciferin to evaluate ATP level. Assays of this type have the underlying limitation that they are measuring cellular metabolism, which is not cellular viability per se. It is quite common for cells to become quiescent under adverse conditions, but still retain the ability to divide3,4,5. For example, cancer stem cells are often relatively metabolically quiescent6,7,8,9, and are likely to be difficult to assay using these techniques. Effectiveness of treatments that damage mitochondrial function, such as most mitocans, is also likely to be significantly overestimated.
An alternative methodology leverages the chemical properties of various substances that allow them to either cross or not cross biological membranes. One example are&#160;nuclear stains such as SYTOX or propidium iodide (PI). This category also includes assays that are similar in concept but different in function, such as the lactate dehydrogenase (LDH) assay, which measures the release of LDH into the extracellular milieu as an indicator of cellular necrosis (Figure 1, Table 1). These assays are more capable of distinguishing between metabolically inactive and dead cells.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Assay/dye</td>
			<td>Type(s) of cell death detected</td>
			<td>Necessary equipment</td>
			<td>Key features</td>
		</tr>
		<tr>
			<td>MTT, CKK-8, Alamar Blue (resazurin)</td>
			<td>Apoptosis/Necrosis</td>
			<td>Spectrophotometer</td>
			<td>Inexpensive, rapid; endpoint assay; dependent on enzymes&#39; activity (exclusively mitochondrial in case of MTT) and does not discriminate between modes of cell death1,10</td>
		</tr>
		<tr>
			<td>LDH release</td>
			<td>Necrosis</td>
			<td>Spectrophotometer</td>
			<td>Rapid, independent of mitochondrial enzymes&#8217; activity; expensive for high-throughput tests; detects necrotic cells with compromized plasma membrane11,12</td>
		</tr>
		<tr>
			<td>Trypan Blue (TB)</td>
			<td>Apoptosis/Necrosis</td>
			<td>Microscope</td>
			<td>Cell-impermeant; does not discriminate between modes of cell death; laborous and not suitable for high-throughput screening; more difficult to use with adherent cells; prone to subjective judgment of the user, but is considered the standard cell viability measurement method13</td>
		</tr>
		<tr>
			<td rowspan="2">Acridine orange (AO)</td>
			<td>Apoptosis/Necrosis/</td>
			<td rowspan="2">Fluorescence microscope</td>
			<td rowspan="2">A nucleic acid dye with unique spectral properties, can distinguish between apoptosis and necrosis/necroptosis14</td>
		</tr>
		<tr>
			<td>Necroptosis</td>
		</tr>
		<tr>
			<td>Hoechst 33342, DAPI</td>
			<td>Apoptosis</td>
			<td>Fluorescence microscope or flow cytometer</td>
			<td>Cell-permeable; inappropriate on its own to monitor cell death; useful for co-staining; can be used to assess chromatin condensation and nuclei fragmentation in early apoptosis; can be paired with propidium iodide to distinguish apoptosis from necrosis15,16</td>
		</tr>
		<tr>
			<td>Propidium Iodide (PI)</td>
			<td>Late apoptosis/Necrosis</td>
			<td>Fluorescence microscope or flow cytometer</td>
			<td>Cell-impermeant intercalator; detects both late apoptosis and necrosis modes of cell death17. Toxic and permeable after long incubation times18</td>
		</tr>
	</tbody>
</table>
Table 1. List of cytotoxicity assays. Cytotoxicity assays, some of which were used in this study, listed along with the brief description of their key features.
Recent studies have demonstrated that mitochondrial metabolism is altered in some cancers19,20,21,22,23,24,25. For example, acute myeloid leukemias (AML)&#160;have been shown to upregulate their mitochondrial mass, mtDNA content, and mitochondrial respiration to meet their energy demands19,26,27. On the other hand, some solid tumors are characterized by mitochondrial dysfunction, or rather &#8220;metabolic reprogramming&#8221;, such as downregulation of mitochondrial proteins involved in OXPHOS or decreased mtDNA content, that has been associated with tumor invasiveness, metastatic potential and resistance to apoptosis-inducing drugs28,29. Furthermore, recently, there has been an increased interest in using mechanistically diverse compounds that affect mitochondrial function (generally called mitocans30), as potential therapies for particular cancers. These drugs target ATP synthesis, mitochondrial DNA, OXPHOS, and ROS production, as well as pro-apoptotic and anti-apoptotic proteins associated with mitochondria30,31. Several studies have shown that this approach has significant promise19,32,33,34. However, these metabolic deviations in cancer cell biology or mitochondria-targeting treatments may significantly affect conventional viability assays that are based on mitochondrial functionality.
Here, an optimized protocol for a differential nuclear staining assay is described. The protocol allows fast and accurate determination of cytotoxicity of mitocans or their combinations with other compounds. Hoechst 33342 is a cell-permeant nuclear dye that readily crosses cell membranes to stain DNA, allowing the total cell count to be obtained. By co-staining with PI, which only enters the nuclei of dead cells, the proportion of living (Hoechst only) and dead (stained with both) cells can be accurately determined. This protocol refines the published assay35 by adding a step for the optimization of the dye concentration (by cross-referencing results with orthogonal trypan blue method) and centrifugation of the plate prior to imaging. Since many cell lines are semi-adherent or suspended, centrifugation increases the proportion of cells that are imaged and strongly improves accuracy. The assay has several advantages, including that staining does not require removal of media or washing. The dye mixture is also inexpensive, easy to prepare, and compatible with multichannel/robotic pipetting systems.
After cells have been stained, they are imaged with an automated microscope. This has the added advantage of creating a permanent record of the images that can be re-analyzed later and the effects of particular compounds can be re-evaluated by visual inspection of captured images. Once images have been obtained, cells can be counted either manually or by using any of several software packages, including both free (e.g., ImageJ, CellProfiler, etc) and commercial software (e.g., Metamorph, Gen5, etc). Automated cell counting is generally preferable since properly developed automated cell counting pipelines are more accurate and less biased than manual counts. They also more effectively disregard cell debris or insoluble complexes. Development of these pipelines is generally straightforward and is simplified by the efficiency of the stains used. The output is quantitative since the actual number of dead cells is automatically calculated with respect to total cell number, and different thresholds can be applied to increase or decrease the stringency of detection35. For convenience, optimized parameters for counting cells using Gen5 v. 3.00 software compatible Cytation 5 Cell Imaging Multi-Mode Reader&#160;are included.

<table border="0" cellpadding="0" cellspacing="0" style="width:1423px;" width="1423">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">2-Deoxy-D-glucose/2-DG</td>
			<td style="width:137px;">Chem-Impex</td>
			<td style="width:176px;">50-519-067</td>
			<td style="width:819px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">3-bromo-pyruvate</td>
			<td>Alfa Aesar</td>
			<td style="width:176px;">1113-59-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">96-Well plates</td>
			<td style="width:137px;">Greiner Bio-One</td>
			<td style="width:176px;">655090</td>
			<td>Black or clear flat-bottomed 96-well plates 
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alamar blue HS cell viability reagent (100mL)&#160;</td>
			<td style="width:137px;">Thermo Fisher</td>
			<td>A50101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Countess II automated cell counter</td>
			<td style="width:137px;">Thermo Fisher</td>
			<td style="width:176px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytation 5 Cell Imaging Multi-Mode Reader</td>
			<td style="width:137px;">BioTek</td>
			<td style="width:176px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Hoechst 33342</td>
			<td style="width:137px;">Thermo Fisher</td>
			<td style="width:176px;">62249</td>
			<td>20 mM solution; final concentration 1:1,000 
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HyClone fetal bovine serum</td>
			<td>GE Healthcare</td>
			<td>#25-514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">m-chlorophenylhydrazone/CCCP</td>
			<td>Sigma Aldrich</td>
			<td style="width:176px;">C2759</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">PBS tablets</td>
			<td style="width:137px;">Thermo Fisher</td>
			<td>BP2944100</td>
			<td>1 tablet + 200 mL of sterile water = 1x PBS solution 
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin-Glutamine (100X)</td>
			<td>Gibco</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Pierce LDH assay kit</td>
			<td style="width:137px;">Thermo Fisher</td>
			<td>50-103-5952</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Propidium Iodide</td>
			<td style="width:137px;">Thermo Fisher</td>
			<td>50-596-072</td>
			<td>Dry powder; stock 1 mg/mL in PBS; final concentration 5 &#181;g/mL (leukemia cells), 1 &#181;g/mL (normal PBMCs) 
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Rotenone</td>
			<td>Ark Pharm</td>
			<td style="width:176px;">AK115691</td>
			<td></td>
		</tr>
		<tr height="83">
			<td height="83" style="height:84px;width:291px;">RPMI-1640 Medium 
			With L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture</td>
			<td style="width:137px;">Sigma Aldrich</td>
			<td style="width:176px;">R8758-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiazolyl blue tetrazolium bromide</td>
			<td style="width:137px;">ACROS Organics</td>
			<td style="width:176px;">AC158990010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Trypan blue stain (0.4%)</td>
			<td style="width:137px;">Gibco</td>
			<td style="width:176px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1423px;">Cell lines</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">K562</td>
			<td style="width:137px;">ATCC</td>
			<td style="width:176px;">CCL-243</td>
			<td>CML cell line 
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">MOLM-13</td>
			<td style="width:137px;">ATCC</td>
			<td style="width:176px;"></td>
			<td>AML cell line 
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">MOLT-4</td>
			<td style="width:137px;">ATCC</td>
			<td>CRL-1582</td>
			<td>ALL cell line 
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">OCI-AML2</td>
			<td style="width:137px;">ATCC</td>
			<td style="width:176px;"></td>
			<td>AML cell line 
			</td>
		</tr>
	</tbody>
</table>

an automated differential nuclear staining assay for accurate determination of mitocan cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The aforementioned protocol has been developed using OCI-AML2 cells, which were taken as a representative acute myeloid leukemia&#160;cell line. AML is characterized by abnormal proliferation of undifferentiated and non-functional hematopoietic cells in the bone marrow26. Despite recent developments in AML targeted therapy, the standard of care has remained unchanged for several decades, and consists of induction therapy (typically comprised of three days of anthracycline, e.g., daunorubicin, idar...

Watch this Scientific Journal Video about An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity at JoVE.com

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

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