Nick Asbrock1, Konstantin Taganov1, Vi Chu1, Martin G. Pomper2, Il Minn2, Haofan Wang2, Steven D. Leach2
1Assay & Platform Development, Bioscience BU, MilliporeSigma, 28820 Single Oak Drive, Temecula, CA, USA 9259, 2Johns Hopkins Russel H. Morgan Department of Radiology and Radiological Science, Baltimore, Maryland 21287, USA.
Cancer stem cells (CSCs) are subpopulations of cancer cells that can self-renew, generate diverse cells in the tumor mass, and sustain tumorigenesis. Some researchers believe that cancer arises from cancer stem cells that originate as a result of mutational hits on normal stem cells. High ALDH activity serves as a universal marker of stem cells, both normal and malignant. Cells can be identified and isolated based upon the enzymatic activity of ALDH, a detoxifying enzyme responsible for oxidation of hazardous aldehyde byproducts. The marker ALDH has been used to isolate cancer stem cells from various human malignancies including bladder, breast, cervical, colon, head and neck, liver, lung, pancreas, prostate and ovary. Historically, selection of cells positive for aldehyde dehydrogenase (ALDH) activity within a green fluorescent background has been difficult with existing reagents.
The AldeRed™ ALDH Detection Kit provides cancer and stem cell scientists with new capabilities for live cell isolation and characterization. The AldeRed™ reagent is a red-shifted fluorescent substrate for aldehyde dehydrogenase (ALDH), allowing cancer stem cells to be identified and isolated by flow cytometry with concurrent use of green fluorescent cell lines, antibodies, transgenic animals and reporter assays.
Pomper MG et al. A red-shifted fluorescent substrate for aldehyde dehydrogenase. Nat Commun. 2014 Apr 23;5:3662.
Figure 1. AldeRed™ Mechanism of Action. AldeRed™ 588-A is a fluorescent and non-toxic ALDH substrate that diffuses freely into intact and viable cells, but remains trapped inside the cells once converted by ALDH into the corresponding acid. The amount of fluorescence produced is proportional to the ALDH activity in the cells and is measured by flow cytometry, allowing fluorescence-activated cell sorting (FACS). For optimal excitation and emission of AldeRed™ 588-A, use a combination of blue laser (488 nm) or green laser (532 nm) with PE-Texas Red detector (615 nm).
AldeRed™ Assay Buffer Preparation
AldeRed™ 588-A Preparation
Note: For optimal excitation and emission of AldeRed™ 588-A, use a combination of blue laser (488 nm) or green laser (532 nm) with PE-Texas Red detector (615 nm). Detection in proximal measurement channels (e.g. PE) is also possible, but may affect delta MFI (mean fluorescence intensity) between Test sample and DEAB treated control sample.
Figure 2. ALDEFLUOR™ and AldeRed™ identify similar CSC populations. ALDH detection in SKBR3 cells using the ALDEFLUOR™ assay yielded 91% ALDHhi population of cells using a combination of Blue laser (488 nm) with FITC detection (525 nm). ALDH detection in SKBR3 cells using the AldeRed™ assay yielded 92% ALDHhi population of cells using a combination of Green laser (532 nm) with PE-Texas red detector (615 nm).
Figure 3. AldeRed™ allows for the co-labeling of CSC populations with FITC-conjugated antibodies. Co-staining of SK-BR-3 cells with AldeRed™ 588-A and Milli-Mark™ EpCam-FITC antibody (clone mAB8 Cat. No. FCMAB264F) yields 54% ALDHhiEpCAMhi.
Figure 4. ALDH CSC identification in Head and Neck Squamous Carcinoma cell lines. UM-SCC-47 is a unique head and neck squamous carcinoma cell line isolated from primary tumor and containing integrated human papillomavirus (HPV-16) and characterized by presence of ALDH+ cancer stem cell sub-populations. A) ALDH detection in UM-SCC-47 cancer cell line by AldeRed™ 588-A yields 13% ALDHhi CSC populations. B) Effect of signaling pathway inhibitors on ALDHHi CSC populations. UM-SCC-47 cells were treated for 24 hrs with 1 uM of each inhibitor and ALDH activity was measured by AldeRed™ 588-A. Notably, AKT Inhibitor IV (124011) and EGFR inhibitor (324674) increased the overall ALDH positive CSC populations.
Figure 5. Confocal images of CA/TD murine pancreatic cells co-stained with ALDEFLUOR™ and AldeRed™ 588-A.
ALDEFLUOR™ is a registered trademark of Aldagen Inc.
AldeRed™ is a trademark of Merck KGaA, Darmstadt, Germany and/or its affiliates.