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 medium. For example, if testing compounds dissolved in DMSO and methanol, make solvent-only control for each solvent.</li>
	</ol>
	</li>
	<li>Collect cells from culture dish or flask into a 15 mL conical tube.</li>
	<li>Transfer 10 &#181;L of cell suspension into a microcentrifuge tube and stain with 10 &#181;L&#160;0.4% trypan blue. Use a hemocytometer to count viable and non-viable cells for each cell source.</li>
	<li>Pellet cells at 200&#160;g for 5 min. Aspirate or decant supernatant.</li>
	<li>Resuspend cell pellet in assay-appropriate media (serum-free or 1, 2.5, 5% FBS RPMI-1640) at a cell density of 3*105 cells/mL. 
	NOTE&#160;1: Cell density of 3*105 cells/mL provides a seeding density of 15,000 cells/well. Seeding density is an important parameter and ideally should be pre-defined prior to the experiment. Seeding density should take into account 1) cell size &#8211; usually larger cells are seeded at a lower density; 2) treatment duration &#8211; cells are typically seeded at a lower density for experiments that will last longer; and 3) cell division rate &#8211; cells with a higher rate of division are seeded at a lower density. Specific examples of optimized seeding densities: K562 cells, larger, 24 h duration &#8211; 10,000 cells/well; MOLM-13 cells, moderate size, 24 h treatment &#8211; 15,000/well; MOLM-13 cells, 48 h treatment &#8211; 8,000/well; small healthy peripheral blood mononuclear cells (PBMCs), 24 h treatment &#8211; 50,000/well; primary AML cells, 24 h treatment &#8211; 15,000-20,000/well. 
	NOTE&#160;2:&#160;The presence of FBS in media may affect the activity of the compounds. Reducing the concentration of FBS may make assay results simpler to interpret, but it also reduces the physiological accuracy.</li>
	<li>Seed 50 &#181;L of cell suspension from step 1.5 into each well of a 96-well plate using a multichannel pipette.</li>
	<li>Add compounds as follows:
	<ol>
		<li>For single compound assays, add 50 &#181;L of 2x compound solution into each well. For solvent-control wells, add 50 &#181;L of test media containing the solvent at the 2x concentration.</li>
		<li>For combination assays, add 25 &#181;L of each of the compound (4x solutions) into each well. For single compound-control wells, add 25 &#181;L of 4x compound solution and 25 &#181;L of test medium. For solvent-control wells, add 50 &#181;L of test medium or test medium containing the solvent. 
		NOTE&#160;1: The final concentration of DMSO should not exceed 0.5%. 
		NOTE&#160;2:&#160;It is recommended to add the media containing the compounds with the pipette touching the wall of each well due to low volume.</li>
	</ol>
	</li>
	<li>Gently tap the plate to ensure mixing of the contents of the wells.</li>
	<li>Incubate plates at 37 &#176;C in a humidified 5% CO2 atmosphere for an appropriate time, e.g., 24 h.</li>
</ol>
2. Cytotoxicity Assay: staining with Hoechst 33342 and propidium Iodide
<ol>
	<li>Prepare 10x staining solution. This solution needs to be prepared fresh before each experiment,&#160;it cannot be stored. Final dye concentrations should be determined prior to the experiment.
	<ol>
		<li>For leukemia cell lines and primary leukemia cells, 1 mL of 10x staining buffer contains&#160;10 &#181;L of 20 mM Hoechst 33342 and 50 &#181;L of 1 mg/mL propidium iodide in sterile PBS (final concentrations: Hoechst 33342 20 &#181;M, PI 5 &#181;g/mL).</li>
		<li>For healthy PBMCs, 1 mL of 10x staining buffer contains&#160;10 &#181;L of 20 mM Hoechst 33342 and 10 &#181;L of 1 mg/mL propidium iodide in sterile PBS (final concentrations: Hoechst 33342 20 &#181;M, PI 1 &#181;g/mL). 
		NOTE: The final concentration of propidium iodide has to be determined prior to the experiments. Cells should be tested using a range of PI concentrations (1, 2.5, 5 &#181;g/mL), and then Hoechst/PI-calculated viability should be compared with viability measured via trypan blue. The PI concentrations listed above were chosen based on target cell viability in media-control wells (above ~90% for leukemia cell lines, above ~70% for healthy&#160;PBMCs). 
		CAUTION: Hoechst 33342 and propidium iodide are potential carcinogens. Wear appropriate personal protective equipment when handling them.</li>
	</ol>
	</li>
	<li>After incubation, use a multichannel pipette to add 10 &#181;L of 10x staining buffer to each well. 
	NOTE: To prevent cross-contamination, make sure that the pipette tips do not touch the media.</li>
	<li>Gently tap the plate to mix and clear bubbles. Stain at 37 &#176;C for 15 min.</li>
	<li>Centrifuge the plate at 200&#160;g for 4 min to bring all of the cells to the bottom of the plate. Carefully wipe the bottom of the plate with a damp kimwipe&#160;to remove fibers and/or debris that will interfere with imaging. 
	NOTE 1: Centrifugation of the plate ensures the highest chances for all of the cells to be captured in the image. Often dead cells will detach and float, providing misleading values of cytotoxicity. Centrifugation mitigates this effect. 
	NOTE 2:&#160;The plate should be imaged as quickly as possible after centrifugation, ideally, within 15 min. Centrifugation can reduce the selectivity of PI staining and may allow cells to slowly accrue propidium iodide. The improvement in accuracy gained by visualizing the dead cells outweighs the slight increase in PI staining. It is recommended to finish imaging within 1 h of centrifugation.</li>
</ol>
3. Cytotoxicity Assay: data acquisition
<ol>
	<li>Set the software for the automated microscope/plate imager to detect fluorescence for both Hoechst 33342 (excitation maximum 350 nm, emission maximum 461 nm) and PI (excitation maximum 493 nm, emission maximum 636 nm). Acquire images for each well in both channels.</li>
	<li>Using the software (such as CellProfiler, a free image analysis studio based on MatLab http://cellprofiler.org/, ImageJ, or proprietary software such as Gen5) count the cells in each well in each channel. 
	NOTE: Since every cell should be stained with Hoechst 33342, and dead cells should be&#160;stained with PI, the ratio of dead to all represents the fraction of cells that are dead. For example, if the automated count in untreated sample shows 467 cells stained with PI (dead cells) and 2335 cells stained with Hoechst 33342 (total cells), the fraction dead is 0.2 or 20%. This value is then compared to an identically handled sample where treatment was used.</li>
	<li>Detailed description of Hoechst/PI&#160;data acquisition using Cytation5 Cell Imaging Multi-Mode Reader and Gen5 v. 3.00 software:
	<ol>
		<li>Set the Cytation5 multimode plate reader/imager to image cells in flat bottom, 96-well, generic black plastic plates.</li>
		<li>Set an imaging protocol to utilize the standard DAPI and Texas Red filter sets. Take images at the well center using a 4x magnification objective. Use no offset (X/Y or Z). Use the following imaging settings: DAPI &#8211; LED - 10, integration time - 99, gain - 0; Texas Red &#8211; LED - 8, integration time - 950, gain - 18. Perform autofocusing using the DAPI signal; there should be no offset in focusing between channels.</li>
		<li>Perform image analysis using Gen5 software v 3.00. In the software settings, define cells as shapes between 5 and 25 &#181;m in their size. Exclude primary edge objects and split touching objects by turning on special option &#8220;Split touching objects&#8221;.&#160; Next, process the image to remove the background (dark background subtraction), apply a nuclear mask (threshold value DAPI &#62;= 6000 AU), and count objects. Perform a subpopulation analysis based on PI staining (threshold value Texas Red &#62;= 5000 AU), and count objects. Cell viability % is defined as&#160;&#160;(1 - <img alt="Equation 1" src="/files/ftp_upload/61295/61295eq1.jpg" />.</li>
	</ol>
	</li>
</ol>

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<li>Tunçer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low+dose+dimethyl+sulfoxide+driven+gross+molecular+changes+have+the+potential+to+interfere+with+various+cellular+processes%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Low dose dimethyl sulfoxide driven gross molecular changes have the potential to interfere with various cellular processes.</a> Scientific Reports. 8 (1), 14828(2018).</li>
</ol>

The authors have nothing to disclose.

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 progression39,40.
Second, the staining should be performed as soon as possible after treatment. Since there are no washing steps, the compound remains in the wells and can continue to inflict damage on the cells. In comparison to the earlier protocol35, the cells were stained for only 15 minutes. This limits the possibility that viable cells could be inadvertently stained with propidium iodide. For this reason, we suggest staggering treatments and staining if more than two plates will be treated. This allows for imaging time between plates and improves reproducibility.
Third, appropriate dye concentration is dependent on cell type. It is important to empirically determine the optimized propidium iodide concentration, beginning with untreated cells, using trypan blue staining as an orthogonal reference.
Finally, the centrifugation step immediately before visualization is also critical. Based on this protocol, a range of 500-4,000 cells is recorded (depending on seeding density). This is a notable improvement over the 100-400 cells per well used previously35. Considering the variation in the population of cancer cells (especially primary cells), having more cells for analysis is important, and may allow more robust data analysis.
Due to the relative simplicity of the method, a wide variety of modifications can easily be performed. For example, many other cell-impermeant dyes are available from a number of vendors and can be substituted for propidium iodide. While this substitution has relatively little value on its own, the increased palette of colors means that more complex experiments can be performed. For example, the addition of a third color will allow differential survival to be assessed between subpopulations of cells, as is commonly performed with flow cytometry.
A more substantial modification of the protocol is foregoing cell visualization and using a spectrophotometer instead. This approach has the advantage of utilizing a nearly ubiquitous piece of equipment (a standard spectrophotometer with a microplate reader) and is the fastest method to acquire data. However, this method is far less accurate. Fluorescence intensity is representative of cell staining, but stochastic variations in staining intensity, combined with the relatively smaller sample area, introduce significant variability. While further optimization (such as the inclusion of a fixative, additional washing steps, or a larger number of samples) may address these problems, fluorescence microscopy is generally a superior approach.
In conclusion, the modified Hoechst/PI protocol is a fast, accurate, inexpensive, high-throughput cytotoxicity assay that is independent of mitochondrial function. This assay has substantial utility for efficiently screening compounds or compound combinations that target mitochondria.

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><tbody><tr><td>2-Deoxy-D-glucose/2-DG</td><td>Chem-Impex</td><td>50-519-067</td><td></td></tr><tr><td>3-bromo-pyruvate</td><td>Alfa Aesar</td><td>1113-59-3</td><td></td></tr><tr><td>96-Well plates</td><td>Greiner Bio-One</td><td>655090</td><td>Black or clear flat-bottomed 96-well plates&lt;br/&gt; </td></tr><tr><td>Alamar blue HS cell viability reagent (100mL)&amp;nbsp;</td><td>Thermo Fisher</td><td>A50101</td><td></td></tr><tr><td>Countess II automated cell counter</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Cytation 5 Cell Imaging Multi-Mode Reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>Hoechst 33342</td><td>Thermo Fisher</td><td>62249</td><td>20 mM solution; final concentration 1:1,000&lt;br/&gt; </td></tr><tr><td>HyClone fetal bovine serum</td><td>GE Healthcare</td><td>#25-514</td><td></td></tr><tr><td>m-chlorophenylhydrazone/CCCP</td><td>Sigma Aldrich</td><td>C2759</td><td></td></tr><tr><td>PBS tablets</td><td>Thermo Fisher</td><td>BP2944100</td><td>1 tablet + 200 mL of sterile water = 1x PBS solution&lt;br/&gt; </td></tr><tr><td>Penicillin-Streptomycin-Glutamine (100X)</td><td>Gibco</td><td>10378016</td><td></td></tr><tr><td>Pierce LDH assay kit</td><td>Thermo Fisher</td><td>50-103-5952</td><td></td></tr><tr><td>Propidium Iodide</td><td>Thermo Fisher</td><td>50-596-072</td><td>Dry powder; stock 1 mg/mL in PBS; final concentration 5 &amp;micro;g/mL (leukemia cells), 1 &amp;micro;g/mL (normal PBMCs)&lt;br/&gt; </td></tr><tr><td>Rotenone</td><td>Ark Pharm</td><td>AK115691</td><td></td></tr><tr><td>RPMI-1640 Medium&lt;br/&gt; With L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture</td><td>Sigma Aldrich</td><td>R8758-500ML</td><td></td></tr><tr><td>Thiazolyl blue tetrazolium bromide</td><td>ACROS Organics</td><td>AC158990010</td><td></td></tr><tr><td>Trypan blue stain (0.4%)</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>&lt;strong&gt;Cell lines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>K562</td><td>ATCC</td><td>CCL-243</td><td>CML cell line&lt;br/&gt; </td></tr><tr><td>MOLM-13</td><td>ATCC</td><td></td><td>AML cell line&lt;br/&gt; </td></tr><tr><td>MOLT-4</td><td>ATCC</td><td>CRL-1582</td><td>ALL cell line&lt;br/&gt; </td></tr><tr><td>OCI-AML2</td><td>ATCC</td><td></td><td>AML cell line&lt;br/&gt; </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, idarubicin, or anthracenedione mitoxanthrone,&#160;and 7 days of cytarabine) followed by consolidation (typically comprised of rounds of cytarabine treatment followed by periods of recovery)36.
Following the protocol outlined in Figure 1A, OCI-AML2 cells were seeded in 96-well plates and treated with the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) at a range of concentrations. Cells were treated for 24 h at 37 &#176;C in serum-free RPMI-1640 media, and then viability was assessed using trypan blue (TB) exclusion or one of four viability assays (Hoechst/PI differential nuclear staining, LDH release assay, MTT, or alamarBlue). Representative images of cells stained with Hoechst/PI are shown in Figure 2A. Several important observations can be made immediately. First, the total number of cells (Hoechst staining) is reasonably high and is markedly greater than the number of dead cells (PI staining). This suggests that the media conditions are not triggering high rates of cell death. Second, since only cells being labeled with both Hoechst and PI are counted as dead (purple cells in the merged image), the probability of counting debris is very low. This image shows a good example of properly stained cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61295/61295fig1.jpg" /> 
Figure 1. Experimental timeline and comparison of existing cytotoxicity assays. (A) Flowchart summarizing the timeline for the experimental procedure, e.g. Hoechst/PI staining. (B) Comparison of cytotoxicity assays, some of which were used in this study. Dye exclusion assays involve impermeant nuclear dyes that stain dead cells with compromised plasma membrane: TB &#8211; trypan blue, PI &#8211; propidium iodide, EtBr &#8211; ethidium bromide, and SYTOX. Second group of assays depend on cellular metabolism, for example, tetrazolium salts MTT, XTT, and CKK-8 (WST-8), resazurin-based reagent alamarBlue, etc. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61295/61295fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61295/61295fig2.jpg" /> 
Figure 2. Comparison of a panel of cytotoxicity assays with trypan blue exclusion. (A) Representative images of total (Hoechst 33342) and dead (propidium iodide, PI) OCI-AML2 cells via Hoechst/PI staining. (B) Assessment of viability of OCI-AML2 cells using different methods after treatment with a gradient of CCCP (top) or 2-DG (bottom) concentrations. OCI-AML2 were treated with CCCP or 2-DG in serum-free RPMI-1640 for 24 h prior to determination of cell viability. Shown is mean, error bars represent SEM. C. Difference in cell viability between cytotoxicity assays in (B) vs.&#160;trypan blue staining (see Supplementary Tables S1-2 for the exact numbers and median difference). Stars indicate significant difference vs. trypan blue staining. ** p &#60; 0.01, *** p &#60; 0.001, ns &#8211; non-significant. Group comparison was done via t-test with correction for multiple hypothesis testing. Three independent biological replicates were performed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61295/61295fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As we recently reported19, leukemias are very sensitive to mitotoxic treatments, which indicates that the cells already have underlying mitochondrial damage. On this basis, we predicted that the MTT and alamarBlue assays, which are based on mitochondrial enzyme activity, would inaccurately measure cellular viability. As expected, these assays (especially alamarBlue) showed significantly lower viability compared to trypan blue exclusion, see Figure 2B,C, Supplementary Tables S1-S2). This is consistent with the fact that doses of these compounds required to induce apoptosis or necrosis are higher than those needed to compromise mitochondrial function.
Amongst the assays that were tested, dual staining with Hoechst 33342 and PI had the best combination of robustness, sensitivity, and consistency with TB staining, with the smallest median deviation from TB exclusion method after CCCP or 2-DG treatment (Supplementary Tables S1-2). Interestingly, at higher doses of CCCP (50 and 100 &#181;M) viability estimated with Hoechst/PI or TB was increased compared to lower doses (Figure 2B, top). This is likely due to the precipitation of CCCP at higher doses due to its hydrophobicity, reducing its effective concentration and impact on cells. Increasing the dose of CCCP further reduced Hoechst/PI-estimated mean viability of OCI-AML2 cells, however: 74% at 150 &#181;M, 62% at 200 &#181;M, 6% at 300 &#181;M (data not shown).
The Hoechst/PI assay was also effective at determining cellular viability after treatment with other mitochondria-targeting molecules. These included rotenone, a poison that compromises Complex I of the mitochondrial electron transport chain37, and 3-bromopyruvate, a glycolytic inhibitor and alkylating agent that impairs mitochondrial respiration and mitochondrial metabolism38 (Figure 3A-D, Supplementary Tables S3-4).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61295/61295fig3.jpg" /> 
Figure 3. Validation of Hoechst/PI cytotoxicity assay in leukemia cells. (A,B) OCI-AML2 (AML) cells were treated with different concentrations of rotenone (A) or 3-bromopyruvate, 3-BP (B) in serum-free RPMI-1640 media for 24 h, then viability was determined. Shown is mean with SEM. (C,D) Comparison of viability difference between Hoechst/PI assay vs.&#160;trypan blue staining (for the cells in A-B, see Supplementary Tables S3-4 for quantitation). Stars indicate statistical significance vs.&#160;trypan blue staining. ** p &#60; 0.01, *** p &#60; 0.001, ns &#8211; non-significant. Group comparison was done via t-test with correction for multiple hypothesis testing. E. MOLM-13 (AML), primary AML cells isolated from a patient, MOLT-4 (ALL), and K562 (CML) cells were treated with the indicated concentrations of rotenone in serum-free RPMI-1640 media for 24 h, prior to viability determination using Hoechst/PI staining. Shown is mean with SEM. Three independent biological replicates were performed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61295/61295fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The Hoechst/PI cytotoxicity assay was further validated using a panel of leukemia cell lines representing multiple types of leukemia, as well as primary cells. They included MOLM-13 (an AML cell line), primary AML cells derived from a representative patient sample, MOLT-4 (an acute lymphoblastic leukemia cell line, ALL), and K562 (a chronic myelogenous leukemia cell line, CML) (Figure 3E, Supplementary Figure S1). Results of the Hoechst/PI assay showed that these cells had profound differences in rotenone sensitivity, ranging from very sensitive (MOLT-4) to resistant (K562) cells.
To demonstrate the robustness of the assay, OCI-AML2 cells were treated with a concentration gradient of 3-bromopyruvate, as described in the protocol above. Cell counts were collected for 6 wells at each concentration and are shown (Figure 4A,B). These counts were used to calculate viability, and showed that as few as 4 wells were sufficient to accurately capture assay outcome (Figure 4C). The resulting dose-response curve is shown (Figure 4D).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61295/61295fig4.jpg" /> 
Figure 4. Reproducibility of Hoechst/PI staining. Viability of OCI-AML2 cells after 24 h of treatment with different concentrations of 3-bromopyruvate (3-BP). (A) Total&#160;number of cells counted via Hoechst 33342 staining, (B) Number of dead cells counted via propidium iodide staining. (C) Viability (%) calculated using (A-B). (D) Representative dose-response graph. Shown is mean with SEM. Three independent biological replicates were performed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61295/61295fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Although the assay is relatively robust, care must still be taken. Improper performance of the assay may compromise its accuracy. Several compromised outcomes are shown for troubleshooting purposes (Figure 5). The first set of images shows the consequences of over-staining with PI, which may arise from the following sources: using the dye at too high concentration, staining for too long, or using too high LED intensity/integration time in the red channel (Figure 5A). These errors will generate artificially inflated numbers of dead cells. The second issue arises from neglecting the centrifugation step. Often, dead cells begin to lose their attachment from the surface of the dish. Consequently, they will be underrepresented during image acquisition, which generally occurs near the bottom of the well (Figure 5B). Finally, overexposure in the Hoechst channel artificially expands the size of the cells, significantly reducing the total counts per well and limiting the assay power (Figure 5C).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61295/61295fig5.jpg" /> 
Figure 5. Comparison of optimal and sub-optimal assay parameters. Comparison of results acquired via optimized parameters (left) or sub-optimal parameters (right). (A) Healthy&#160;PBMCs were stained with propidium iodide at 1 &#181;g/mL (left) or 5 &#181;g/mL (right) for 15 min before imaging. (B) Images of OCI-AML2 cells taken with (left) or without (right) plate centrifugation. (C) Images of OCI-AML2 cells acquired with optimal (left) or excessive (right) integration time for the Hoechst channel. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61295/61295fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">Difference with trypan blue method, %</td>
		</tr>
		<tr>
			<td>CCCP, &#181;M</td>
			<td>Hoechst/PI</td>
			<td>LDH assay</td>
			<td>MTT</td>
			<td>Alamar Blue</td>
		</tr>
		<tr>
			<td>3.1</td>
			<td>-1.19</td>
			<td>33.00</td>
			<td>-31.05</td>
			<td>-59.04</td>
		</tr>
		<tr>
			<td>6.3</td>
			<td>6.99</td>
			<td>41.90</td>
			<td>-11.03</td>
			<td>-59.34</td>
		</tr>
		<tr>
			<td>12.5</td>
			<td>0.71</td>
			<td>41.60</td>
			<td>4.31</td>
			<td>-52.52</td>
		</tr>
		<tr>
			<td>25</td>
			<td>30.73</td>
			<td>66.33</td>
			<td>-3.68</td>
			<td>-51.85</td>
		</tr>
		<tr>
			<td>50</td>
			<td>47.38</td>
			<td>50.71</td>
			<td>-17.71</td>
			<td>-60.27</td>
		</tr>
		<tr>
			<td>100</td>
			<td>13.81</td>
			<td>17.12</td>
			<td>-45.31</td>
			<td>-67.25</td>
		</tr>
		<tr>
			<td>Median</td>
			<td>10.40</td>
			<td>41.75</td>
			<td>-14.37</td>
			<td>-59.19</td>
		</tr>
	</tbody>
</table>
Supplementary Table S1. Viability differences for a panel of assays in OCI-AML2 cells after CCCP treatment. Assessment of viability was performed after 24 h of treatment with different concentrations of CCCP. Median viability differences between tested assays (Hoechst/PI, LDH, MTT, or alamarBlue) and trypan blue staining with automated counting was calculated based on viability differences at each drug concentration.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">Difference with trypan blue method, %</td>
		</tr>
		<tr>
			<td>2-DG, mM</td>
			<td>Hoechst/PI</td>
			<td>LDH assay</td>
			<td>MTT</td>
			<td>Alamar Blue</td>
		</tr>
		<tr>
			<td>3.1</td>
			<td>16.16</td>
			<td>20.77</td>
			<td>5.82</td>
			<td>-20.27</td>
		</tr>
		<tr>
			<td>6.3</td>
			<td>22.93</td>
			<td>31.96</td>
			<td>-11.13</td>
			<td>-31.11</td>
		</tr>
		<tr>
			<td>12.5</td>
			<td>30.85</td>
			<td>45.07</td>
			<td>-34.27</td>
			<td>-36.29</td>
		</tr>
		<tr>
			<td>25</td>
			<td>13.81</td>
			<td>42.39</td>
			<td>-55.79</td>
			<td>-52.29</td>
		</tr>
		<tr>
			<td>50</td>
			<td>16.96</td>
			<td>81.12</td>
			<td>-29.38</td>
			<td>-47.68</td>
		</tr>
		<tr>
			<td>100</td>
			<td>7.79</td>
			<td>149.16</td>
			<td>-19.37</td>
			<td>-28.67</td>
		</tr>
		<tr>
			<td>Median</td>
			<td>16.56</td>
			<td>43.73</td>
			<td>-24.38</td>
			<td>-33.70</td>
		</tr>
	</tbody>
</table>
Supplementary Table S2. Viability differences for a panel of assays in OCI-AML2 cells after 2-DG treatment. Assessment of viability was performed after 24 h of treatment with different concentrations of 2-DG. Median viability differences between tested assays (Hoechst/PI, LDH, MTT, or alamarBlue) and trypan blue staining with automated counting were calculated based on viability differences at each drug concentration.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Rotenone, &#181;M</td>
			<td>Viability difference, % (Hoechst/PI-trypan blue)</td>
		</tr>
		<tr>
			<td>10</td>
			<td>4.45</td>
		</tr>
		<tr>
			<td>25</td>
			<td>7.61</td>
		</tr>
		<tr>
			<td>50</td>
			<td>17.70</td>
		</tr>
		<tr>
			<td>100</td>
			<td>7.74</td>
		</tr>
		<tr>
			<td>150</td>
			<td>56.38</td>
		</tr>
		<tr>
			<td>200</td>
			<td>16.57</td>
		</tr>
		<tr>
			<td>Median</td>
			<td>12.15</td>
		</tr>
	</tbody>
</table>
Supplementary Table S3. Viability difference between Hoechst/PI and TB exclusion method in OCI-AML2 cells after rotenone treatment. Assessment of viability was performed after 24 h of treatment with different concentrations of rotenone. Median viability difference between Hoechst/PI and trypan blue staining with automated counting was calculated based on viability differences at each drug concentration.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>3-BP, &#181;M</td>
			<td>Viability difference, % (Hoechst/PI-trypan blue)</td>
		</tr>
		<tr>
			<td>5</td>
			<td>5.73</td>
		</tr>
		<tr>
			<td>10</td>
			<td>9.00</td>
		</tr>
		<tr>
			<td>25</td>
			<td>7.72</td>
		</tr>
		<tr>
			<td>50</td>
			<td>-1.90</td>
		</tr>
		<tr>
			<td>100</td>
			<td>-28.23</td>
		</tr>
		<tr>
			<td>200</td>
			<td>-20.77</td>
		</tr>
		<tr>
			<td>Median</td>
			<td>1.91</td>
		</tr>
	</tbody>
</table>
Supplementary Table S4. Viability difference between Hoechst/PI and TB exclusion method in OCI-AML2 cells after 3-bromopyruvate (3-BP) treatment. Assessment of viability was performed after 24 h of treatment with different concentrations of 3-BP. Median viability difference between Hoechst/PI and trypan blue staining with automated counting was calculated based on viability differences at each drug concentration.
Supplementary Figure S1. Representative images of cells stained with Hoechst/PI, with or without rotenone treatment at indicated concentrations in serum-free RPMI-1640 media for 24 h:&#160;A-B.&#160;AML: MOLM-13 cell line (A), and primary AML cells (B) derived from a representative patient sample. C. ALL: MOLT-4 cell line. D. CML: K562 cell line. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61295/61295supfig1large.jpg" target="_blank">Please click here to download this file.</a>

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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Nanyang Technological University

Research

JoVE Journal

Cancer Research

6.8K Views.  Rice University. Deze methode zal onderzoekers in staat stellen om cellulaire levensvatbaarheid onafhankelijk van mitochondriale functie te beoordelen. En het is vatbaar voor hoge doorvoer drug screening inspanningen. Deze techniek is snel, nauwkeurig, goedkoop, en maakt de bepaling van verbindingen cytotoxiciteit, met inbegrip van die afbreuk doen aan mitochondriale functie in de meeste soorten cellen.Dit protocol is eenvoudig uit te voeren. Het is belangrijk om aandacht te besteden aan het geneesmidd...

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

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Immunology and Infection

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Bioengineering

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Biology

Automation of the Micronucleus Assay Using Imaging Flow Cytometry and Artificial Intelligence

The micronucleus (MN) assay is used worldwide by regulatory bodies to evaluate chemicals for genetic toxicity. The assay can be performed in two ways: by scoring MN in once-divided, cytokinesis-blocked binucleated cells or fully divided mononucleated cells. Historically, light microscopy has been the gold standard method to score the assay, but it is laborious and subjective. Flow cytometry has been used in recent years to score the assay, but is limited by the inability to visually confirm key aspects of cellular imagery. Imaging flow cytometry (IFC) combines high-throughput image capture and automated image analysis, and has been successfully applied to rapidly acquire imagery of and score all key events in the MN assay. Recently, it has been demonstrated that artificial intelligence (AI) methods based on convolutional neural networks can be used to score MN assay data acquired by IFC. This paper describes all steps to use AI software to create a deep learning model to score all key events and to apply this model to automatically score additional data. Results from the AI deep learning model compare well to manual microscopy, therefore enabling fully automated scoring of the MN assay by combining IFC and AI.

The micronucleus (MN) assay is a well-established test for quantifying DNA damage. However, scoring the assay using conventional techniques such as manual microscopy or feature-based image analysis is laborious and challenging. This paper describes the methodology to develop an artificial intelligence model to score the MN assay using imaging flow cytometry data.

The micronucleus (MN) assay is used worldwide by regulatory bodies to evaluate chemicals for genetic toxicity. The assay can be performed in two ways: by ...

Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level

The assessment of the cytotoxic potential of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell treatments, is instrumental in assessing their efficacy and is a prerequisite for clinical application. However, traditional cytotoxicity assays are conducted as bulk assays and do not provide detailed information about the functional heterogeneity of the CAR T cell population. In this study, we describe a double-emulsion droplet-based method that allows for large-scale co-encapsulation of single effector CAR T cells with single target cells while enabling dual quantification of both cytotoxic effector molecules from T cells and cell death of the target cell. The protocol outlines a method for the generation and purification of CD19-specific CAR T cells, followed by their co-encapsulation in droplets with the CD19+ cell line JeKo-1, along with reagents to visualize cytotoxic effector molecule secretion (Granzyme B) and cell death (using propidium iodide, PI). We demonstrate how to generate droplets containing single CAR T and target cells using a commercially available microfluidics device for generating double emulsion droplets. Additionally, we provide examples of how to assay the functional diversity of CAR T cells in droplets using standard flow cytometry equipment. Finally, we briefly describe the temporal kinetics and heterogeneity of CD19-specific CAR T-cell killing. While this method focuses on cell death following a CAR T cell attack, it is also adaptable for examining other types of T cells, cytotoxic immune cells, and effector cell functions, such as cytokine secretion.

Here, we describe a method for creating double-emulsion droplets encapsulating one T cell and a cancer target cell to examine cell killing at the single-cell level. This method allows for dual quantification of both cytotoxic molecules and target-cell apoptosis within a large population of T cells.

The assessment of the cytotoxic potential of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell treatments, is instrumental in ...

Measuring Mitochondrial Function of Na&#239;ve and Effector CD8 T Cells

Understanding how immunometabolism impacts the function, differentiation, and fate of lymphocytes has garnered significant interest and attention. Lymphocyte biology has been explored using bioenergetic analysis and has now become a critically import tool in the field. Thus, we sought to optimize a bioenergetic analysis assay that can be adapted with pretreatments and acute injection for receptor stimulations. Here, we evaluated CD8 T cell ex vivo metabolism using the Cell Mito Stress Test to assess rates of oxygen consumption and extracellular acidification in na&#239;ve and effector CD8 T cells. Antigen-specific effector CD8 T cells were derived via ex vivo stimulation, and na&#239;ve CD8 T cells harvested from splenocytes and isolated with magnetic bead column separation. 
Pretreatments are performed in microplates and we detail how to prepare sensor cartridges. We show how injection ports can loaded with drugs to indirectly measure metabolic capacities and with metabolic modulators, this protocol can be used to study specific enzyme activity. T-cell receptor stimulations can be studied in real time with acute injection and stimulation with anti-CD3/CD28 using the injection ports. Instrument analyzers are used for measurements and data collection and data visualization is done with software programs to interpret cellular metabolism. This strategy produces an extensive amount of data on immune cell biology and mitochondrial bioenergetics allowing researchers to customize the protocol in numerous ways to explore CD8 T cell metabolism.

CD8 T cell bioenergetics can be interrogated using the Mito Stress Test. This methodology can be used to study acute and chronic metabolic programming. This protocol describes approaches to examine the relationships between T cell receptor biology and bioenergetic analysis.

Understanding how immunometabolism impacts the function, differentiation, and fate of lymphocytes has garnered significant interest and attention. ...

An In Vitro Assay to Study the Cytotoxic Capability of Cytokine-Induced Killer Cells

Source: Hsiao, C. H., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60420">Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment.</a> J. Vis. Exp. (2020).
This video demonstrates an in vitro technique to measure the cytotoxic ability of cytokine-induced killer (CIK) cells against cancer cells. Upon co-culturing target cancer cells with CIK cells, the dead cancer cells are identified using a fluorescent viability dye and are quantified using flow cytometry, estimating the cytotoxic effect of CIK cells.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Differential Nuclear Staining Assay: An Assay to Determine Mitocan Cytotoxicity by Propidium Iodide and Hoechst Double Staining

Source: Pei, J. et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/61295">An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity.</a> J. Vis. Exp. (2020)
This video demonstrates a nuclear staining method to detect the cytotoxic effect of mitocan on drug-sensitive cancer cells and healthy variants. The double staining with DNA-binding dyes such as propidium iodide and Hoechst dye provides a clear distinction between the dead and live cells, thus helping to assess the cytotoxicity of the drug.

Source: Pei, J. et al., An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity. J. Vis. Exp. (2020)
This ...

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

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