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 ...

This method will allow researchers to assess cellular viability independently of mitochondrial function. And it is amenable to high throughput drug screening efforts. This technique is fast, accurate, inexpensive, and allows the determination of compounds cytotoxicity, including those impairing mitochondrial function in most cells types.This protocol is easy to perform. It is important to pay attention to the drug preparation, treatment conditions, and cell density to ensure experimental accuracy and validity. Visual demonstration of this method is important because image acquisition and image analysis steps can be hard to recapitulate, especially for researchers having little experience with Gen5 Software.Begin by preparing solutions of the compounds of interest at the desired concentration. Always make sure to mix compounds by vortexing thoroughly. To measure cytotoxicity of a single compound, prepare compounds at 2X final concentration.If measuring cytotoxicity of compound combinations, prepare them at a 4X final concentration. Prepare solvent only controls by mixing the same amount of solvent with the appropriate medium. Collect cells into a 15 milliliter conical tube and aliquot 10 microliters of the cell suspension.For trypan blue staining, add 10 microliters of 0.4%trypan blue to the aliquot and use a hemocytometer or cell counter to count viable and non-viable cells. Pellet cells in the 15 milliliter tube at 200 times G for five minutes, then aspirate or decamped the supernatant. We suspend the cell pellet in essay appropriate media at a cell density of three times 10 to the fifth cells per milliliter.Use a multi-channel pipette to seed 50 microliters of cell suspension into each well of a 96 well plate. For single compound assays, add 50 microliters of 2X compound solution into each well. For solvent control wells, add 15 microliters of test media containing the solvent at 2X concentration.For combination assays add 25 microliters of each of the 4X compounds into each well. For single compound control wells, add 25 microliters each of 4X compound solution and test medium. Make sure that the final concentration of DMSO does not exceed 0.5%To ensure the reproducibility, add drugs with the pipette tips touching the side of wells.Being careful to add the drugs to different locations. This stabs should be performed very quickly, preferably taking no longer than 30 minutes per plate. Gently tap the plate to mix the contents of the wells and incubate it at 37 degrees Celsius and 5%carbon dioxide.When ready to stain the cells with Hoechst 33342 and propidium iodide prepare fresh 10X staining solution and add 10 microliters to each well. Gently tap the plate and incubate it at 37 degrees Celsius for 15 minutes. Centrifuge the plate at 200 times G for four minutes to bring all the cells to the bottom of the plate.Then carefully wipe the bottom of the plate with a damp laboratory wipe to remove any debris that may interfere with imaging. Image the plate within 15 minutes after centrifugation. Open Gen5 Software and create a new standard protocol, click on Procedure and choose the type of plates to be used.Usually, 96 well black plastic plates. Next, set the read method to image, click Okay, and choose DAPI and Texas Red Filter sets with a 4X magnification objective. No offset is needed, since the well centers will be imaged.For the DAPI filter, set LED to 10, integration time to 99, and gain to zero. For Texas Red, set LED to eight, integration time to 950, and gain to 18. Click on Options to make sure that the auto focus is performed on the DAPI channel and there is no offset in focusing between channels.Save the protocol by clicking Okay. Now images can be recorded using the Read New button. Once images are recorded, click on Data Reduction and choose Image Preprocessing.Apply Image Preprocessing with dark background subtraction using Auto flattening size based on the DAPI signal. For Texas Red, use the same options as for Channel 1. When finished, click Okay.Then go to the cellular analysis panel and apply a Nuclear Mask based on the transformed DAPI signal with a threshold value of 6, 000 units, minimal object size of five micrometers and maximal object size of 25 micrometers. Analyze the entire image, exclude primary edge objects on the border of the image and split touching objects. In advanced detection options, set the rolling ball diameter as 30 micrometers, and evaluate the background based on 5%of lowest pixels.Only keep the cell count in calculated metrics. After that, perform a subpopulation analysis based on the mean transformed Texas Red signal with a threshold value of 5, 000 units to count dead cells. Finally, access the resulting cell counts.Representative images of cells stained with Hoechst, propidium iodide are shown here. The total number of cells is reasonably high and greater than the number of dead cells. When a panel of cytotoxicity assays was compared with trypan blue exclusion, it was found that MTT and the Alamar blue assays inaccurately measured cellular viability.These mitochondrial enzyme-based assays showed significantly lower viability compared to trypan blue exclusion. Dual staining with Hoechst 33342 and PI had the best combination of robustness, sensitivity, and consistency with trypan blue staining and the smallest median deviation from the trypan blue exclusion method after CCP or 2-DG treatment. The Hoechst PI essay was also effective at determining cellular viability after treatment with the mitochondria targeting molecules Rotenone and three bromopyruvate.It was further validated with a panel of leukemia cell lines. These cells varied in Rotenone sensitivity ranging from very sensitive to resistance cells. Improper performance of the assay may compromise its accuracy.Several compromised outcomes are shown here. Overstaining with PI, neglecting the centrifugation step, or overexposure in the Hoechst channel, will cause suboptimal results. While timing this protocol, remember to optimize the dye concentration and staining time for each cell line.Also, centrification for a sample plate, is necessary to ensure imaging quality. After identification of compounds of cytotoxicity effect on cancer cells, researchers may proceed with mechanistic studies to identify their relevant targets. Screening small molecule libraries with this technique in parallel to conventional MTT assays, will allow molecules that target mitochondrial function to be rapidly identified.

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Research

JoVE Journal

Cancer Research

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes through their two different arms. bsAbs have been actively studied for their ability to directly recruit immune cells to kill tumor cells. Currently, the majority of bsAbs are produced in the form of recombinant proteins, either as Fc-containing bsAbs or as smaller bsAb derivatives without the Fc region. In this study, GPC3-S-Fab, an antibody fragment (Fab) based bispecific antibody, was designed by linking the Fab of anti-GPC3 antibody GC33 with an anti-CD16 single domain antibody. The GPC3-S-Fab can be expressed in Escherichia coli and purified by two affinity chromatographies. The purified GPC3-S-Fab can specifically bind to and kill GPC3 positive liver cancer cells by recruiting natural killer cells, suggesting a potential application of GPC3-S-Fab in liver cancer therapy.

Here, we present a protocol to produce a bispecific antibody GPC3-S-Fab in Escherichia coli. The purified GPC3-S-Fab has potent cytotoxicity against GPC3 positive liver cancer cells.

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. Although heterogeneous in presentation, a common denominator among these tumors is the overexpression of somatostatin receptors. 68Ga-DOTATATE is a somatostatin analog labeled with the positron emitter gallium-68 (68Ga). For well-differentiated neuroendocrine tumors, 68Ga-DOTATATE positron emission tomography (PET)/computed tomography (CT) imaging is used for diagnosis, determination of disease burden, and therapy selection.
This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiolabeling of 68Ga-DOTATATE is performed with a fully automated labeling module coupled to a germanium-68 (68Ge)/68Ga generator. Quality control of the final product evaluates radiochemical purity with instant thin-layer chromatography and solid-phase chromatography, and pH prior to patient injection. Periodic quality control is performed to determine 68Ge breakthrough, sterility, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) content. Patient preparation includes patient instructions, a protocol for 68Ga-DOTATATE during treatment with somatostatin analogs, and intravenous administration of the radiopharmaceutical. For PET/CT imaging, the acquisition and reconstruction settings are described. For each step, radiation safety will be highlighted, as well as time constrictions due to the short half-life of 68Ga.
Fully automated in-house production and quality control of 68Ga-DOTATATE leads to very high success rates (95%) and produces two to four patient dosages per batch, depending on the yield of the generator. In conclusion, 68Ga-DOTATATE PET/CT imaging is a noninvasive and fast method of providing information on the tumor burden of neuroendocrine tumors (NETs) while also assisting in diagnosis and therapy selection.

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Well-differentiated neuroendocrine tumors overexpress somatostatin receptors which can be utilized for diagnostic imaging with the radiolabeled somatostatin analog 68Ga-DOTATATE. This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiation safety and time constrictions due to the short half-life of 68Ga are taken into account.

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Management of Respiratory Motion Artefacts in 18F-fluorodeoxyglucose Positron Emission Tomography using an Amplitude-Based Optimal Respiratory Gating Algorithm

Positron emission tomography (PET) combined with X-ray computed tomography (CT) is an important molecular imaging platform that is required for accurate diagnosis and clinical staging of a variety of diseases. The advantage of PET imaging is the ability to visualize and quantify a myriad of biological processes in vivo with high sensitivity and accuracy. However, there are multiple factors that determine image quality and quantitative accuracy of PET images. One of the foremost factors influencing image quality in PET imaging of the thorax and upper abdomen is respiratory motion, resulting in respiration-induced motion blurring of anatomical structures. Correction of these artefacts is required for providing optimal image quality and quantitative accuracy of PET images.
Several respiratory gating techniques have been developed, typically relying on acquisition of a respiratory signal simultaneously with PET data. Based on the respiratory signal acquired, PET data is selected for reconstruction of a motion-free image. Although these methods have been shown to effectively remove respiratory motion artefacts from PET images, the performance is dependent on the quality of the respiratory signal being acquired. In this study, the use of an amplitude-based optimal respiratory gating (ORG) algorithm is discussed. In contrast to many other respiratory gating algorithms, ORG permits the user to have control over image quality versus the amount of rejected motion in the reconstructed PET images. This is achieved by calculating an optimal amplitude range based on the acquired surrogate signal and a user-specified duty cycle (the percentage of PET data used for image reconstruction). The optimal amplitude range is defined as the smallest amplitude range still containing the amount of PET data required for image reconstruction. It was shown that ORG results in effective removal of respiration-induced image blurring in PET imaging of the thorax and upper abdomen, resulting in improved image quality and quantitative accuracy.

Management of Respiratory Motion Artefacts in 18F-fluorodeoxyglucose Positron Emission Tomography using an Amplitude-Based Optimal Respiratory Gating Algorithm

Amplitude-based optimal respiratory gating (ORG) effectively removes respiratory-induced motion blurring from clinical 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) images. Correction of FDG-PET images for these respiratory motion artefacts improves image quality, diagnostic and quantitative accuracy. Removal of respiratory motion artefacts is important for adequate clinical management of patients using PET.

Positron emission tomography (PET) combined with X-ray computed tomography (CT) is an important molecular imaging platform that is required for accurate ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Three Differential Expression Analysis Methods for RNA Sequencing: limma, EdgeR, DESeq2

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration and complex biological processes and has great value in diagnostics, prognostics, and therapeutics of tumors. Differential analysis of RNA-seq data is crucial to identify aberrant transcriptions, and limma, EdgeR and DESeq2 are efficient tools for differential analysis. However, RNA-seq differential analysis requires certain skills with R language and the ability to choose an appropriate method, which is lacking in the curriculum of medical education.
Herein, we provide the detailed protocol to identify differentially expressed genes (DEGs) between cholangiocarcinoma (CHOL) and normal tissues through limma, DESeq2 and EdgeR, respectively, and the results are shown in volcano plots and Venn diagrams. The three protocols of limma, DESeq2 and EdgeR are similar but have different steps among the processes of the analysis. For example, a linear model is used for statistics in limma, while the negative binomial distribution is used in edgeR and DESeq2. Additionally, the normalized RNA-seq count data is necessary for EdgeR and limma but is not necessary for DESeq2.
Here, we provide a detailed protocol for three differential analysis methods: limma, EdgeR and DESeq2. The results of the three methods are partly overlapping. All three methods have their own advantages, and the choice of method only depends on the data.

A detailed protocol of differential expression analysis methods for RNA sequencing was provided: limma, EdgeR, DESeq2.

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration ...

An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, &#947;-H2AX. The use of an automated microscopy platform to score the number of &#947;-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the &#947;-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive &#947;-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

An Automated Microscopic Scoring Method for the &amp;#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

This protocol presents the slide preparation and automated scoring of the &#947;-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects ...