A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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.

figure-introduction-3775
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. Please click here to view a larger version of this figure.

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.

Protocol

1. Measure total ALDH1A1 activity in ovarian cancer cell homogenates via fluorimetry

  1. Thaw 1 × 106 cells in a T25 cell culture flask in 5 mL of the following cell culture media:
    1. IGROV-1 and PEO4: Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).
    2. BG-1 and Caov-3: Dulbecco's Modified Eagle's Medium (DMEM) with 10% FBS and 1% P/S.
    3. OVCAR-3: RPMI 1640 with 20% FBS, 1% P/S, and 0.01 mg/mL insulin.
  2. Maintain the cells in an incubator at 37 °C and 5% CO2 for two to three passages. Ensure the cells do not exceed 80%-90% confluency before passaging.
  3. Trypsinize the cells in 0.25% trypsin for 10 min, count them using an automated cell counter, and pellet 1 × 107 cells via centrifugation (180 × g) at 25 °C for 5 min.
  4. Remove the supernatant carefully via aspiration, wash the pellet by resuspending the cells in 1 mL of 1x PBS, and re-pellet the cells via centrifugation under the same conditions as described in step 4.
  5. Resuspend the cells in a solution of 1x protease inhibitor in 1x PBS. Use 1 mL of this solution per 2.5 × 106 cells.
  6. Sonicate the cell suspension on ice for 2 min (1 s pulse, 40% amplitude) with a cell homogenizer probe.
  7. Pellet insoluble/membrane fractions via centrifugation (3,200 × g) at 25 °C for 15 min. Remove and keep the supernatant, as this is the homogenate that will be used in subsequent steps. Separate the homogenates into three cuvettes to perform the experiment in triplicate.
  8. Add the probe to the homogenate to obtain a final probe concentration of 4 µM. Pipette up and down three times to mix the solution well.
  9. Measure the fluorescent signal immediately after addition and after desired incubation times on a fluorimeter. The incubation time can be optimized to see the maximum fold turn-on (1 h in this experiment). The incubation time must be constant across all cell lines compared.
    1. Set the excitation wavelength to 496 nm.
    2. Set the emission to 510-600 nm.
    3. Set the slit width to 0.5 mm.
    4. Pipette the solution into a 1 mL quartz cuvette, place into the fluorimeter, and hit scan.
  10. Divide the final fluorescence intensity at 516 nm (maximum fluorescent wavelength) by the intensity at the same wavelength from the initial reading to determine the fold activation of the isoform-selective dye.

2. Use of fluorescence microscopy to image cells with high ALDH1A1 activity

  1. Thaw 1 × 106 cells in a T25 cell culture flask in 5 mL of the appropriate cell culture media.
  2. Maintain the cells in an incubator at 37 °C and 5% CO2 for two to three passages. Ensure the cells do not exceed 80%-90% confluency before passaging.
  3. The day prior to confocal imaging, plate 4 × 105 cells in an 8-well chamber slide.
    1. Coat the bottom of each well with poly-L-lysine (0.1 mg/mL, 100 µL per well) for 10 min, subsequently aspirating.
    2. Wash each well 3x by adding cell culture grade water (100 µL per well) and aspirating.
    3. Trypsinize the cells in 0.25% trypsin for 10 min, count them using an automated cell counter, and plate the cells at 4 × 105 cells per well.
    4. Let the cells settle and attach overnight (12-16 h).
  4. Aspirate the growth media and add serum-free media (500 µL per well), supplemented with 2 µM of the probe or the control probe.
  5. Incubate with the probe at room temperature for 30 min and image the cells immediately.
  6. Turn on the confocal microscope and adjust for the sample.
    1. Load the sample carefully at the lowest magnification; find the cells to ensure proper positioning.
    2. Ensure the objective lens is at 10x magnification and locate the cells.
  7. For this experiment, the FITC channel (excitation: 488 nm laser; emission: 516-521 nm) and T-PMT (transmitted light) channel are required. Locate and focus on the cells using T-PMT to focus on the same z-plane for the entirety of the experiment to remove bias.
  8. Adjust the laser power and FITC gain to the appropriate setting, where the signal from the Ctrl-AlDeSense AM samples is minimally detectable, while still seeing signal in AlDeSense AM samples. Each parameter can be adjusted 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.
  9. Snap three images per well for a total of three wells per treatment condition (nine images in total). Focus on the proper plane using T-PMT to avoid bias rather than using the fluorescence channel or merged image.
  10. Process the images to determine the percentage of ALDH1A1+ cells.
    1. Using image processing software, split the czi file into different channels.
    2. Count the total number of cells and the total number of fluorescent cells.
    3. To determine the percentage of ALDH1A1+ cells, divide the number of fluorescent cells by the total number of cells in each image. It is imperative to count the same way for each image without manipulating the images, as, for instance, adjusting the brightness may add a confounding variable.

3. Application of flow cytometry to identify cells with high ALDH1A1 activity

  1. Thaw 1 × 106 cells in a T25 cell culture flask in 5 mL of the appropriate cell culture media.
  2. Maintain the cells in an incubator at 37 °C and 5% CO2 for two to three passages. Ensure the cells do not exceed 80%-90% confluency before passaging.
  3. Trypsinize, count, and pellet the cells in a 15 mL centrifuge tube via centrifugation (180 × g) at 25 °C for 5 min.
  4. Resuspend the cells in 1 mL of 2 µM probe/control probe solution in PBS. Rock the cells at room temperature for 60 min to ensure exposure to the dye is even.
  5. After the incubation period, pellet the cells via centrifugation (180 × g) at 25 °C for 5 min. Resuspend the cells in 0.5 mL of PBS. Run the cells through a cell strainer (35 µm nylon mesh) to remove cell clumps that may clog the flow cytometer. Immediately place the cells on ice.
  6. Turn the instrument on and run the start-up protocol.
  7. Check for sheath fluid and empty waste.
  8. Run the lines with 10% bleach and water for 5 min each.
  9. Run quality control beads to ensure proper function.
  10. In the settings tab, select FSC (forward scatter), SSC (side scatter), and FITC (fluorescein isothiocyanate) for the fluorescence filter.
  11. Draw the following graphs to gate for viability, singlets, and fluorescence. Optimize the laser power (specific to user's instrument) so that the cell populations are within the given parameters for further analysis.
    1. FSC-A versus SSC-A scatter plot: main cell population is near the center of the graph.
    2. FSC-A versus FSC-W scatter plot: narrow horizontal band indicative of singlets (rather than cell clumps).
    3. FITC-A versus FSC-A scatter plot: observe the distribution of cells sorted by the isoform-selective fluorogenic turn-on probe.
    4. FITC-A histogram: observe the shift in population based on FITC to determine the percentage of ALDH1A1+ cells.
  12. 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-A signal. Subsequently, run a sample with the control probe. A population shift should be observable to reveal the maximum dynamic range. 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.
  13. Run each sample for 10,000 counts (done in triplicate).
  14. Repeat step 3.12 for each cell line, as there will be variability in uptake and ALDH1A1 activity.
  15. After completion of sample collection, run the lines with 10% bleach and water for 5 min each, then initiate shutdown of the instrument.
  16. Process the data using the flow cytometry software and gate the desired cell population. Set the gates so that all events fall into either the ALDH1A1- or ALDH1A1+ gate. Using the rectangle gate selection, set the ALDH1A1- gate so that >99.5% of events in the control probe samples occur within this gate. The remaining cells will be considered ALDH1A1+. These same gates can then be applied to the probe sample to quantify the number of events considered ALDH1A1- and ALDH1A1+.

Results

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 ± 0.01); IGROV-1 (1.30 ± 0.03); Caov-3 (1.72 ± 0.06); PEO4 (2.51 ± 0.29); and OVCAR-3 (10.25 ± 1.46) (Figure 2).

figure-results-431
Figure 2<...

Discussion

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...

Disclosures

We disclose a pending patent (US20200199092A1) for the AlDeSense technology.

Acknowledgements

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.

Materials

NameCompanyCatalog NumberComments
 0.25% Trypsin, 0.1% EDTA in HBSS w/o Calcium, Magnesium and Sodium BicarbonateCorning  25-050-CI
1x Phosphate Buffer SalineCorning21-040-CMX12
AccuSpin Micro 17RFisher Scientific13-100-675
AlDeSenseSynthesized in-house
BG-1A gift provided by the Prof. Paul Hergenrother Lab, University of Illinois Urbana-Champaign
BioLite 25cm2 FlaskThermo Fisher Scientific 130189
Biosafety Cabinet 1300 series A2Thermo Fisher Scientific 
Caov-3A gift provided by the Prof. Erik Nelson Lab, University of Illinois Urbana-Champaign
Cell homogenizerFisher Scientific
Centrifuge 5180REppendorf22627040
Contrl-AlDeSenseSynthesized in-house
DMEM, 10% FBS, 1% P/SPrepared by UIUC cell media facility
Falcon Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Cap, 5mLCorning352003
FCS Express 6Provided by UIUC CMtO
FL microscopeEVOS
FluorometerPhoton Technology International
Forma Series II Water-Jacketed CO2 IncubatorFisher Scientific3110
IGROV-1A gift provided by the Prof. Erik Nelson Lab, University of Illinois Urbana-Champaign
ImageJNIH
Innova 42R Incubated Shaker
LSM 700Zeiss
LSR IIBD
Nunc Lab-Tek Chambered #1.0 Borosicilate Coverglass SystemThermo Fisher Scientific 155383
OVCAR-3ATCCHTB-161
PEO4A gift provided by the Prof. Erik Nelson Lab, University of Illinois Urbana-Champaign
Pierce Protease Inhibitor TabletsThermo ScientificA32963
Poly-L-LysineCultrex3438-100-01
RockerVWR
RPMI, 10% FBS, 1% P/SPrepared by UIUC cell media facility
RPMI, 20% FBS, 1% P/S, 0.01 mg/mL InsulinPrepared by UIUC cell media facility

References

  1. Bonnet, D., Dick, J. E. Human acute myeloid leukaemia is organised as a heirarchy that originates from a primitive haematopoetic cell. Nature Medicine. 3 (7), 730-737 (1997).
  2. Begicevic, R. R., Falasca, M. ABC transporters in cancer stem cells: Beyond chemoresistance. International Journal of Molecular Sciences. 18 (11), 2362 (2017).
  3. Cojoc, M., Mäbert, K., Muders, M. H., Dubrovska, A. A role for cancer stem cells in therapy resistance: Cellular and molecular mechanisms. Seminars in Cancer Biology. 31, 16-27 (2015).
  4. Islam, F., Gopalan, V., Smith, R. A., Lam, A. K. Y. Translational potential of cancer stem cells: A review of the detection of cancer stem cells and their roles in cancer recurrence and cancer treatment. Experimental Cell Research. 335 (1), 135-147 (2015).
  5. Li, F., Tiede, B., Massagué, J., Kang, Y. Beyond tumorigenesis: Cancer stem cells in metastasis. Cell Research. 17 (1), 3-14 (2007).
  6. Kim, W. T., Ryu, C. J. Cancer stem cell surface markers on normal stem cells. BMB Reports. 50 (6), 285-298 (2017).
  7. Pors, K., Moreb, J. S. Aldehyde dehydrogenases in cancer: An opportunity for biomarker and drug development. Drug Discovery Today. 19 (12), 1953-1963 (2014).
  8. Jackson, B., et al. Update on the aldehyde dehydrogenase gene (ALDH) superfamily. Human Genomics. 5 (4), 283-303 (2011).
  9. Vasiliou, V., Pappa, A., Petersen, D. R. Role of aldehyde dehydrogenases in endogenous and xenobiotic metabolism. Chemico-Biological Interactions. 129 (1-2), 1-19 (2000).
  10. Tomita, H., Tanaka, K., Tanaka, T., Hara, A. Aldehyde dehydrogenase 1A1 in stem cells and cancer. Oncotarget. 7 (10), 11018-11032 (2016).
  11. Anorma, C., et al. Surveillance of cancer stem cell plasticity using an isoform-selective fluorescent probe for aldehyde dehydrogenase 1A1. ACS Central Science. 4 (8), 1045-1055 (2018).
  12. Bearrood, T. E., Aguirre-Figueroa, G., Chan, J. Rational design of a red fluorescent sensor for ALDH1A1 displaying enhanced cellular uptake and reactivity. Bioconjugate Chemistry. 31 (2), 224-228 (2020).
  13. Chan, J., Dodani, S. C., Chang, C. J. Reaction-based small-molecule fluorescent probes for chemoselective bioimaging. Nature Chemistry. 4 (12), 973-984 (2012).
  14. East, A. K., Lucero, M. Y., Chan, J. New directions of activity-based sensing for in vivo NIR imaging. Chemical Science. 12 (10), 3393-3405 (2021).
  15. Yadav, A. K., et al. Activity-based NIR bioluminescence probe enables discovery of diet-induced modulation of the tumor microenvironment via nitric oxide. ACS Central Science. 8 (4), 461-472 (2022).
  16. Yadav, A. K., et al. An activity-based sensing approach for the detection of cyclooxygenase-2 in Live Cells. Angewandte Chemie. 59 (8), 3307-3314 (2020).
  17. Ueno, T., et al. Rational principles for modulating fluorescence properties of fluorescein. Journal of the American Chemical Society. 126 (43), 14079-14085 (2004).
  18. Tanaka, F., Mase, N., Barbas 3rd, C. F. Design and use of fluorogenic aldehydes for monitoring the progress of aldehyde transformations. Journal of the American Chemical Society. 126 (12), 3692-3693 (2004).
  19. Thurber, G. M., Schmidt, M. M., Wittrup, K. D. Antibody tumor penetration: transport opposed by systemic and antigen-mediated clearance. Advanced Drug Delivery Reviews. 60 (12), 1421-1434 (2008).
  20. Marcato, P., Dean, C. A., Giacomantonio, C. A., Lee, P. W. K. Aldehyde dehydrogenase its role as a cancer stem cell marker comes down to the specific isoform. Cell Cycle. 10 (9), 1378-1384 (2011).
  21. Meng, E., et al. ALDH1A1 maintains ovarian cancer stem cell-like properties by altered regulation of cell cycle checkpoint and DNA repair network signaling. PLoS One. 9 (9), e107142 (2014).
  22. Kaipio, K., et al. ALDH1A1-related stemness in high-grade serous ovarian cancer is a negative prognostic indicator but potentially targetable by EGFR/mTOR-PI3K/aurora kinase inhibitors. The Journal of Pathology. 250 (2), 159-169 (2020).
  23. Deng, S., et al. Distinct expression levels and patterns of stem cell marker, aldehyde dehydrogenase isoform 1 (ALDH1), in human epithelial cancers. PLoS One. 5 (4), e10277 (2010).
  24. Chang, B., et al. ALDH1 expression correlates with favorable prognosis in ovarian cancers. Modern Pathology. 22 (6), 817-823 (2009).
  25. Gardner, S. H., Reinhardt, C. J., Chan, J. Advances in activity-based sensing probes for isoform-selective imaging of enzymatic activity. Angewandte Chemie. 60 (10), 5000-5009 (2021).
  26. Reid, B. M., Permuth, J. B., Sellers, T. A. Epidemiology of ovarian cancer: a review. Cancer Biology and Medicine. 14 (1), 9-32 (2017).
  27. Tulake, W., et al. Upregulation of stem cell markers ALDH1A1 and OCT4 as potential biomarkers for the early detection of cervical carcinoma. Oncology Letters. 16 (5), 5525-5534 (2018).
  28. Nwani, N. G., et al. A novel ALDH1A1 inhibitor targets cells with stem cell characteristics in ovarian cancer. Cancers. 11 (4), 502 (2019).
  29. Roy, M., Connor, J., Al-Niaimi, A., Rose, S. L., Mahajan, A. Aldehyde dehydrogenase 1A1 (ALDH1A1) expression by immunohistochemistry is associated with chemo-refractoriness in patients with high-grade ovarian serous carcinoma. Human Pathology. 73, 1-6 (2018).
  30. Landen Jr, C. N., et al. Targeting aldehyde dehydrogenase cancer stem cells in ovarian cancer. Molecular Cancer Therapeutics. 9 (12), 3186-3199 (2010).
  31. Condello, S., et al. β-catenin-regulated ALDH1A1 is a target in ovarian cancer spheroids. Oncogene. 34 (18), 2297-2308 (2015).
  32. Storms, R. W., et al. Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity. Proceedings of the National Academy of Sciences. 96 (16), 9118-9123 (1999).
  33. Duellman, S. J., et al. A bioluminescence assay for aldehyde dehydrogenase activity. Analytical Biochemistry. 434 (2), 226-232 (2013).
  34. Minn, I., et al. A red-shifted fluorescent substrate for aldehyde dehydrogenase. Nature Communications. 5, 3662 (2014).
  35. Dollé, L., Boulter, L., Leclercq, I. A., van Grunsven, L. A. Next generation of ALDH substrates and their potential to study maturational lineage biology in stem and progenitor cells. American Journal of Physiology. Gastrointestinal and Liver Physiology. 308 (7), 573-578 (2015).
  36. Yagishita, A., et al. Development of highly selective fluorescent probe enabling flow-cytometric isolation of ALDH3A1-positive viable cells. Bioconjugate Chemistry. 28 (2), 302-306 (2017).
  37. Maity, S., et al. Thiophene bridged aldehydes (TBAs) image ALDH activity in cells: Via modulation of intramolecular charge transfer. Chemical Science. 8 (10), 7143-7151 (2017).
  38. Koenders, S. T. A., et al. Development of a retinal-based probe for the profiling of retinaldehyde dehydrogenases in cancer cells. ACS Central Science. 5 (12), 1965-1974 (2019).
  39. Oe, M., et al. Deep-red/near-infrared turn-on fluorescence probes for aldehyde dehydrogenase 1A1 in cancer stem cells. ACS Sensors. 6 (9), 3320-3329 (2021).
  40. Yagishita, A., Ueno, T., Tsuchihara, K., Urano, Y. Amino BODIPY-based blue fluorescent probes for aldehyde dehydrogenase 1-expressing cells. Bioconjugate Chemistry. 32 (2), 234-238 (2021).
  41. Okamoto, A., et al. Identification of breast cancer stem cells using a newly developed long-acting fluorescence probe, C5S-A, targeting ALDH1A1. Anticancer Research. 42 (3), 1199-1205 (2022).

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

Aldehyde Dehydrogenase 1A1ALDH1A1 ActivityOvarian Cancer CellsAlDeSense ApplicationStem Cell BiomarkerFlow CytometryLive Cell ImagingFluorogenic ProbeConfocal Microscope ImagingImage Processing SoftwareFluorescent CellsCell Culture ProtocolCell DetachmentAutomated Cell Counter

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Contact Us

Recommend to library

JoVE NEWSLETTERS

Research

Education

Authors

Librarians

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved