Name: application of aldesense to stratify ovarian cancer cells based on aldehyde dehydrogenase 1a1 activity
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Description: Watch this Scientific Journal Video about Application of AlDeSense to Stratify Ovarian Cancer Cells Based on Aldehyde Dehydrogenase 1A1 Activity at JoVE.com

This work was supported by The National Institutes of Health (R35GM133581 to JC) and the Cancer Center at Illinois Graduate Scholarship (awarded to SG). JC thanks the Camille and Henry Dreyfus Foundation for support. The authors thank Dr. Thomas E. Bearrood for his initial contribution to preparing stocks of AlDeSense and AlDeSense AM. We thank Mr. Oliver D. Pichardo Peguero and Mr. Joseph A. Forzano for their assistance with preparing various synthetic precursors. We thank Prof. Erik Nelson (Department of Molecular and Integrative Physiology, UIUC) for Caov-3, IGROV-1, and PEO4 cells. We thank Prof. Paul Hergenrother (Department of Chemistry, UIUC) for BG-1 cells. We thank the Core Facilities at the Carl R. Woese Institute for Genomic Biology for access to the Zeiss LSM 700 Confocal Microscope and corresponding software. We thank the Flow Cytometry Facility for access to BD LSR II CMtO Analyzer. We thank Dr. Sandra McMasters and the Cell Media Facility for assistance in preparing cell culture media.

1. Measure total ALDH1A1 activity in ovarian cancer cell homogenates via fluorimetry
<ol>
	<li>Thaw 1 &#215; 106 cells in a T25 cell culture flask in 5 mL of the following cell culture media:
	<ol>
		<li>IGROV-1 and PEO4: Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).</li>
		<li>BG-1 and Caov-3: Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with 10% FBS and 1% P/S.</li>
		<li>OVCAR-3: RPMI 1640 with 20% ...

<ol>
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G., 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=A+novel+ALDH1A1+inhibitor+targets+cells+with+stem+cell+characteristics+in+ovarian+cancer.">A novel ALDH1A1 inhibitor targets cells with stem cell characteristics in ovarian cancer.</a> Cancers. 11 (4), 502 (2019).</li><li>Roy, M., Connor, J., Al-Niaimi, A., Rose, S. L., Mahajan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aldehyde+dehydrogenase+1A1+(ALDH1A1)+expression+by+immunohistochemistry+is+associated+with+chemo-refractoriness+in+patients+with+high-grade+ovarian+serous+carcinoma.">Aldehyde dehydrogenase 1A1 (ALDH1A1) expression by immunohistochemistry is associated with chemo-refractoriness in patients with high-grade ovarian serous carcinoma.</a> Human Pathology. 73, 1-6 (2018).</li><li>Landen Jr, C. N., 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=Targeting+aldehyde+dehydrogenase+cancer+stem+cells+in+ovarian+cancer.">Targeting aldehyde dehydrogenase cancer stem cells in ovarian cancer.</a> Molecular Cancer Therapeutics. 9 (12), 3186-3199 (2010).</li><li>Condello, 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=&#946;-catenin-regulated+ALDH1A1+is+a+target+in+ovarian+cancer+spheroids.">&#946;-catenin-regulated ALDH1A1 is a target in ovarian cancer spheroids.</a> Oncogene. 34 (18), 2297-2308 (2015).</li><li>Storms, R. W., 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=Isolation+of+primitive+human+hematopoietic+progenitors+on+the+basis+of+aldehyde+dehydrogenase+activity.">Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity.</a> Proceedings of the National Academy of Sciences. 96 (16), 9118-9123 (1999).</li><li>Duellman, S. 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=A+bioluminescence+assay+for+aldehyde+dehydrogenase+activity.">A bioluminescence assay for aldehyde dehydrogenase activity.</a> Analytical Biochemistry. 434 (2), 226-232 (2013).</li><li>Minn, I., 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=A+red-shifted+fluorescent+substrate+for+aldehyde+dehydrogenase.">A red-shifted fluorescent substrate for aldehyde dehydrogenase.</a> Nature Communications. 5, 3662 (2014).</li><li>Doll&#233;, L., Boulter, L., Leclercq, I. A., van Grunsven, L. 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Gastrointestinal and Liver Physiology. 308 (7), 573-578 (2015).</li><li>Yagishita, A., 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=Development+of+highly+selective+fluorescent+probe+enabling+flow-cytometric+isolation+of+ALDH3A1-positive+viable+cells.">Development of highly selective fluorescent probe enabling flow-cytometric isolation of ALDH3A1-positive viable cells.</a> Bioconjugate Chemistry. 28 (2), 302-306 (2017).</li><li>Maity, 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=Thiophene+bridged+aldehydes+(TBAs)+image+ALDH+activity+in+cells:+Via+modulation+of+intramolecular+charge+transfer.">Thiophene bridged aldehydes (TBAs) image ALDH activity in cells: Via modulation of intramolecular charge transfer.</a> Chemical Science. 8 (10), 7143-7151 (2017).</li><li>Koenders, S. T. A., 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=Development+of+a+retinal-based+probe+for+the+profiling+of+retinaldehyde+dehydrogenases+in+cancer+cells.">Development of a retinal-based probe for the profiling of retinaldehyde dehydrogenases in cancer cells.</a> ACS Central Science. 5 (12), 1965-1974 (2019).</li><li>Oe, M., 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=Deep-red/near-infrared+turn-on+fluorescence+probes+for+aldehyde+dehydrogenase+1A1+in+cancer+stem+cells.">Deep-red/near-infrared turn-on fluorescence probes for aldehyde dehydrogenase 1A1 in cancer stem cells.</a> ACS Sensors. 6 (9), 3320-3329 (2021).</li><li>Yagishita, A., Ueno, T., Tsuchihara, K., Urano, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+BODIPY-based+blue+fluorescent+probes+for+aldehyde+dehydrogenase+1-expressing+cells.">Amino BODIPY-based blue fluorescent probes for aldehyde dehydrogenase 1-expressing cells.</a> Bioconjugate Chemistry. 32 (2), 234-238 (2021).</li><li>Okamoto, A., 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=Identification+of+breast+cancer+stem+cells+using+a+newly+developed+long-acting+fluorescence+probe,+C5S-A,+targeting+ALDH1A1.">Identification of breast cancer stem cells using a newly developed long-acting fluorescence probe, C5S-A, targeting ALDH1A1.</a> Anticancer Research. 42 (3), 1199-1205 (2022).</li></ol>

Pan-selectivity is a major limitation of many ALDH probes; however, several isoform-selective examples have recently been reported32,33,34,35,36,37,38,39,40,41. The isoform-selective fluo...

Cancer stem cells (CSCs) are a subpopulation of tumor cells that exhibit stem cell-like properties1. Similar to their non-cancerous counterparts, CSCs possess the extraordinary ability to self-renew and proliferate. Together with other built-in mechanisms, such as the upregulation of ATP-binding cassette transporters, CSCs are often spared from initial surgical debulking efforts, as well as subsequent adjuvant therapy2. Owing to their critical role in treatment resistance3, relapse4, and metastasis5, CSCs have become a priority in cancer research. Although there are a variety of cell surface antigens (e.g., CD133) that can be used to identify CSCs6, leveraging the enzymatic activity of aldehyde dehydrogenases (ALDHs) found in the cytoplasm has emerged as an attractive alternative7. ALDHs are a superfamily of 19 enzymes responsible for catalyzing the oxidation of reactive endogenous and xenobiotic aldehydes to the corresponding carboxylic acid products8.
In general, aldehyde detoxification is crucial in protecting cells from undesirable crosslinking events and oxidative stress that may damage stem cell integrity9. Moreover, the 1A1 isoform controls retinoic acid metabolism, which in turn influences stemness via retinaldehyde signaling10. AlDeSense11,12, a small-molecule activity-based sensing (ABS) probe to selectively detect ALDH1A1 activity, was recently developed. ABS designs achieve analyte detection through a chemical change rather than a binding event, allowing for high selectivity and decreased off-target responses13,14,15,16. The design principle of the isoform-selective fluorogenic probe relies on a donor-photoinduced electron transfer (d-PeT) quenching mechanism17, originating from the aldehyde functional group, which serves to suppress the fluorescent signature of the probe18. Upon ALDH1A1-mediated conversion to the carboxylic acid, radiative relaxation is unlocked to yield a highly fluorescent product. Because d-PeT quenching is never 100% efficient, the residual fluorescence that may lead to possible false positive results was considered when establishing this assay through the development of Ctrl-AlDeSense, a non-responsive reagent with matching photophysical characteristics (e.g., quantum yield) and an identical cytoplasmic staining pattern in cells. When used in tandem, this unique pairing can reliably distinguish cells with high ALDH1A1 activity from those that exhibit low levels via fluorimetry, molecular imaging, and flow cytometry. Several key advantages are associated with the use of isoform-selective activatable dyes over traditional immunohistochemical methods. For instance, CSCs are hypothesized to be buried deep within a tumor, and thus are more accessible to a small molecule relative to large antibodies19. Additionally, the turned-over fluorescent product does not covalently modify any cellular component, meaning it can be readily removed via wash cycles to leave a CSC in an unmodified state. Lastly, the turn-on response only identifies viable cells and functions, much like the MTT assay, owing to its reliance on the NAD+ cofactor.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64713/64713fig01.jpg" /> 
Figure 1: Schematic demonstrating fluorescent turn-on of AlDeSense. The isoform-selective dye is activated by ALDH1A1 and can be used to identify elevated ALDH1A1 activity in ovarian cancer cells via fluorimetry, molecular imaging, and flow cytometry. <a href="https://www.jove.com/files/ftp_upload/64713/64713fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In past work, the isoform-selective fluorogenic probe assay successfully stratified ALDH high (ALDH+) cells from ALDH low (ALDH-) cells in K562 human chronic leukemia cells, MDA-MB-231 human breast cancer cells, and B16F0 murine melanoma cells. This is important because, for many cancer types, high ALDH1A1 protein expression signifies a worse clinical prognosis20. This presumes that elevated levels of ALDH1A1 are indicative of CSCs which can evade treatment, develop resistance, and disseminate throughout the body. However, in the case of ovarian cancer, there are studies reporting the opposite finding (high ALDH1A1 expression is linked to improved patient survival)21,22,23,24. While this may appear contradictory at first glance, expression does not necessarily correlate to enzyme activity, which may be influenced by changes in the tumor microenvironment (e.g., pH flux, oxygen gradients), availability of the NAD+ cofactor or aldehyde substrates, levels of carboxylic acids (product inhibition), and post-translational modifications that can alter enzyme activity25. Additionally, ovarian cancer is divided into five main histological types (high-grade serous, low-grade serous, endometrioid, clear cell, and mucinous), which we hypothesize will feature variable levels of ALDH1A1 activity26. With the goal of investigating ALDH1A1 activity in ovarian tumors, an isoform-selective fluorogenic probe assay was employed to identify ALDH1A1+ populations in a panel of five ovarian cancer cell lines belonging to the different histological types mentioned above. The cell lines tested in this study include BG-1, Caov-3, IGROV-1, OVCAR-3, and PEO4 cells, covering clear cell and serous histotypes. Herein, the versatility and generalizability of the probe was highlighted to identify CSCs for the researchers that seek to perform similar studies in other immortalized cancer cell lines as well as patient samples. The use of AlDeSense will shed light on the biochemical pathways involved in CSC maintenance in complex tissue microenvironments and potentially serve as a clinical tool for determining prognosis and measuring cancer aggressiveness.

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	<tbody>
		<tr>
			<td>&#160;0.25% Trypsin, 0.1% EDTA in HBSS w/o Calcium, Magnesium and Sodium Bicarbonate</td>
			<td>Corning</td>
			<td>&#160;&#160;25-050-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate Buffer Saline</td>
			<td>Corning</td>
			<td>21-040-CMX12</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuSpin Micro 17R</td>
			<td>Fisher Scientific</td>
			<td>13-100-675</td>
			<td></td>
		</tr>
		<tr>
			<td>AlDeSense</td>
			<td></td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>BG-1</td>
			<td></td>
			<td></td>
			<td>A gift provided by the Prof. Paul Hergenrother Lab, University of Illinois Urbana-Champaign</td>
		</tr>
		<tr>
			<td>BioLite 25cm2 Flask</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>130189</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinet 1300 series A2</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Caov-3</td>
			<td></td>
			<td></td>
			<td>A gift provided by the Prof. Erik Nelson Lab, University of Illinois Urbana-Champaign</td>
		</tr>
		<tr>
			<td>Cell homogenizer</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5180R</td>
			<td>Eppendorf</td>
			<td>22627040</td>
			<td></td>
		</tr>
		<tr>
			<td>Contrl-AlDeSense</td>
			<td></td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>DMEM, 10% FBS, 1% P/S</td>
			<td></td>
			<td></td>
			<td>Prepared by UIUC cell media facility</td>
		</tr>
		<tr>
			<td>Falcon Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Cap, 5mL</td>
			<td>Corning</td>
			<td>352003</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS Express 6</td>
			<td></td>
			<td></td>
			<td>Provided by UIUC CMtO</td>
		</tr>
		<tr>
			<td>FL microscope</td>
			<td>EVOS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorometer</td>
			<td>Photon Technology International</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forma Series II Water-Jacketed CO2&#160;Incubator</td>
			<td>Fisher Scientific</td>
			<td>3110</td>
			<td></td>
		</tr>
		<tr>
			<td>IGROV-1</td>
			<td></td>
			<td></td>
			<td>A gift provided by the Prof. Erik Nelson Lab, University of Illinois Urbana-Champaign</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Innova 42R Incubated Shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 700</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSR II</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Lab-Tek Chambered #1.0 Borosicilate Coverglass System</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>155383</td>
			<td></td>
		</tr>
		<tr>
			<td>OVCAR-3</td>
			<td>ATCC</td>
			<td>HTB-161</td>
			<td></td>
		</tr>
		<tr>
			<td>PEO4</td>
			<td></td>
			<td></td>
			<td>A gift provided by the Prof. Erik Nelson Lab, University of Illinois Urbana-Champaign</td>
		</tr>
		<tr>
			<td>Pierce Protease Inhibitor Tablets</td>
			<td>Thermo Scientific</td>
			<td>A32963</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Cultrex</td>
			<td>3438-100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocker</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI, 10% FBS, 1% P/S</td>
			<td></td>
			<td></td>
			<td>Prepared by UIUC cell media facility</td>
		</tr>
		<tr>
			<td>RPMI, 20% FBS, 1% P/S, 0.01 mg/mL Insulin</td>
			<td></td>
			<td></td>
			<td>Prepared by UIUC cell media facility</td>
		</tr>
	</tbody>
</table>

application of aldesense to stratify ovarian cancer cells based on aldehyde dehydrogenase 1a1 activity

Relapse after cancer treatment is often attributed to the persistence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are characterized by their remarkable tumor-initiating and self-renewal capacity. Depending on the origin of the tumor (e.g., ovaries), the CSC surface biomarker profile can vary dramatically, making the identification of such cells via immunohistochemical staining a challenging endeavor. On the contrary, aldehyde dehydrogenase 1A1 (ALDH1A1) has emerged as an excellent marker to identify CSCs, owing to its conserved expression profile in nearly all progenitor cells including CSCs. The ALDH1A1 isoform belongs to a superfamily of 19 enzymes that are responsible for the oxidation of various endogenous and xenobiotic aldehydes to the corresponding carboxylic acid products. Chan et al. recently developed AlDeSense, an isoform-selective "turn-on" probe for the detection of ALDH1A1 activity, as well as a non-reactive matching control reagent (Ctrl-AlDeSense) to account for off-target staining. This isoform-selective tool has already been demonstrated to be a versatile chemical tool through the detection of ALDH1A1 activity in K562 myelogenous leukemia cells, mammospheres, and melanoma-derived CSC xenografts. In this article, the utility of the probe was showcased through additional fluorimetry, confocal microscopy, and flow cytometry experiments where the relative ALDH1A1 activity was determined in a panel of five ovarian cancer cell lines.

Total ALDH1A1 activity of ovarian cancer cell homogenates 
The average fold turn-ons for each cell line obtained from this assay are: BG-1 (1.12 &#177; 0.01); IGROV-1 (1.30 &#177; 0.03); Caov-3 (1.72 &#177; 0.06); PEO4 (2.51 &#177; 0.29); and OVCAR-3 (10.25 &#177; 1.46) (Figure 2).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64713/64713fig02.jpg" /> 
Figure 2<...

Watch this Scientific Journal Video about Application of AlDeSense to Stratify Ovarian Cancer Cells Based on Aldehyde Dehydrogenase 1A1 Activity at JoVE.com

Application of AlDeSense to Stratify Ovarian Cancer Cells Based on Aldehyde Dehydrogenase 1A1 Activity

Methods to measure ALDH1A1 activity in live cells are critical in cancer research due to its status as a biomarker of stemness. In this study, we employed an isoform-selective fluorogenic probe to determine the relative levels of ALDH1A1 activity in a panel of five ovarian cancer cell lines.

Relapse after cancer treatment is often attributed to the persistence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are ...

Our protocol allows users to measure ALDH1A1 activity, a stem cell biomarker using diverse modalities ranging from flow cytometry to live cell molecular imaging. The major advantages of this approach are that probe activation involves the turnaround response characterized by a high signal to background ratio, as well as selective ALDH1A1 isoform detection. Demonstrating this procedure will be Michael Lee, Sarah Gardner, and Rodrigo Tapia Hernandez graduate students from my laboratory.To begin, aspirate the growth media from the cultured cells of interest in eight well chamber slide. Add 500 microliters per well of serum free media supplemented with a two micromolar isoform selective fluorogenic probe or a non-reactive control probe. Incubate the slide at room temperature for 30 minutes.After incubation, proceed to confocal microscope imaging immediately. Locate the cells at 10 x magnification. The FITC channel and transmitted light channels are required for this experiment.Locate and focus the cells using the transmitted light channel to focus on the same Z plane for the entirety of the experiment to remove bias. Adjust the laser power and FITC gain to the appropriate setting where the signal from the control AlDeSense AM samples is minimally detectable, while still seeing signal in AlDeSense AM samples. Adjust each parameter by sliding the corresponding bar.The settings may have to be adjusted a few times to identify the correct parameters. Once optimized, complete the rest of the experiment within that cell line using identical parameters. Snap three images per well for a total of three wells per treatment condition and save the images.Ensure to focus using the transmitted light channel only while switching between planes and wells to avoid bias. Next, using the image processing software split the CZI file into different channels and count the total number of cells and fluorescent cells. To determine the percentage of aldehyde dehydrogenase 1A1 positive cells, divide the number of fluorescent cells by the total number of cells in each image.Count the same way for each image to avoid confounding variables. Take out a T25 cell culture flask containing the cells maintained in the incubator. Add trypsin for cell detachment, and count the cells using an automated cell counter.Then pellet the cells in a 15 milliliter centrifuge tube by centrifugation at 180G for five minutes at 25 degrees Celsius. Re-suspend the cells in one milliliter of a two micromolar probe or control probe solution in PBS. Rock the cells at room temperature for 60 minutes to ensure even exposure to the dye.After the incubation period, pellet the cells by centrifugation at 180 G for five minutes at 25 degrees Celsius. Then re-suspend the cells in 0.5 milliliters of PBS. Run the cells through a 35 micrometer nylon mesh cell strainer onto a tube placed on ice to remove cell clumps that may clog the flow cytometer.Turn the flow cytometer instrument on and run the startup protocol. Check for sheath fluid and empty waist. Run the lines with 10%bleach and water for five minutes each.Then run quality control beads to ensure proper function. In the settings tab, select forward scatter side scatter and FITC for the fluorescence filter. Draw a forward scatter area versus a side scatter area plot for the main cell population near the center of the graph.Next, draw a forward scatter area, versus a forward scatter width plot for the narrow horizontal bands indicating singlets. Then draw an FITC area versus a forward scatter area plot to observe the distribution of cells sorted by the isoform selective fluorogenic turn on probe. Next, draw an FITC area histogram to observe the shift in population based on FITC and determine the percentage of aldehyde dehydrogenase 1A1 positive cells.To optimize the FITC laser power, run a sample with the probe so that the right tail of the histogram curve is near the maximum FITC area signal. The laser power optimization step may have to be repeated multiple times, but the laser power should not be altered across samples, once a setting has been designated for an experiment. Subsequently, add a sample to the sift, and run it with the control probe.A population shift should be observable to reveal the maximum dynamic range. Run each sample for 10, 000 counts done in triplicate. After completion of sample collection, run the lines with 10%bleach and water for five minutes each, then shut down the instrument.Process the data using flow cytometry software and gait the desired cell population. Using the rectangle gate selection, set the aldehyde dehydrogenase 1A1 negative gait, so that more than 99.5%of events in the control probe samples occur within this gate. The remaining cells will be considered aldehyde dehydrogenase 1A1 positive.Apply the same gates to the probe sample. To quantify the number of events considered aldehyde dehydrogenase 1A1 negative and 1A1 positive. The average fold turn-ons for each cell line were determined to quantify the total aldehyde dehydrogenase 1A1 activity.The percentage of aldehyde dehydrogenase 1A1 positive cells was determined in each cell line by tuning the laser power and gain. To minimize the signal in the control AlDeSense AM treated sample the fluorescence signal was optimized in the AlDeSense AM treated cells. By counting the number of aldehyde dehydrogenase A1A positive cells, the percentage of aldehyde dehydrogenase A1A positive cells was determined.With the application of the isoform selective fluorogenic probe, the aldehyde dehydrogenase 1A1 positive cell population was quantified within each cell line by gating for the top 0.5%of the brightest cells within the control AlDeSense AM treated population. Analysis of the panel of ovarian cancer cells has revealed the percentage of aldehyde dehydrogenase 1A1 positive cells. From the obtained results of the tested cancer cell lines, it can be concluded that BG-1 has the lowest aldehyde dehydrogenase 1A1 activity and lowest aldehyde dehydrogenase 1A1 positive population.Additionally, confocal imaging and flow cytometry revealed the largest percentage of aldehyde dehydrogenase 1A1 positive cells in Caov-3 cells. But the cell line's overall activity was only the third highest. Alternatively, OVCAR-3 cells contained the third highest aldehyde dehydrogenase 1A1 positive population, but exhibited the highest overall activity.AlDeSense is a versatile and generalizable probe to identify CSCs in immortalized cell lines and in patient samples. We hope to see it as a prognostic tool in the clinical setting.

Research

JoVE Journal

Cancer Research

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