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ALDEFLUORâ„¢ Kit

For the identification, evaluation, and isolation of stem and progenitor cells expressing high levels of ALDH

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ALDEFLUORâ„¢ Kit

For the identification, evaluation, and isolation of stem and progenitor cells expressing high levels of ALDH

Catalog #
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For the identification, evaluation, and isolation of stem and progenitor cells expressing high levels of ALDH
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Product Advantages


  • Identify viable cells with elevated ALDH activity

  • Use to identify and isolate normal and malignant stem and progenitor cells across a wide variety of tissues

What's Included

  • Dry ALDEFLUORâ„¢ reagent, 50 µg
  • DEAB Reagent, 1.5 mM, in 95% ethanol, 1 mL (Catalog #01705)
  • HCL, 1.5 mL
  • DMSO, 1.5 mL
  • ALDEFLUORâ„¢ Assay Buffer, 2 x 55 mL (Catalog #01702)
  • ALDEFLUORâ„¢ Quick Reference Guide

Overview

Identify and isolate viable cells expressing aldehyde dehydrogenase (ALDH) with the ALDEFLUORâ„¢ Kit. Compared to traditional methods, this assay does not require antibody staining.

High ALDH expression has been reported for normal and cancer precursor cells of various lineages. The ALDEFLUORâ„¢ assay is a widely published non-immunological method for the detection of ALDH-bright (ALDHbr) cells and can be used to detect cancer cells in hematopoietic, mammary, endothelial, mesenchymal, neural, and other tissues.

ALDEFLUORâ„¢ DEAB Reagent and ALDEFLUORâ„¢ Assay Buffer, included with the kit, support the optimal performance of the assay and are also available for purchase individually.

The kit is compatible with standard flow cytometers for performing downstream analysis of ALDHbr cells and with standard cell sorters for further purification and characterization.

View our additional resources to learn more about the ALDEFLUORâ„¢ reagent system.
Cell Type
Brain Tumor Stem Cells, Cancer Cells and Cell Lines, Hematopoietic Stem and Progenitor Cells, Mammary Cells, Mesenchymal Stem and Progenitor Cells, Neural Stem and Progenitor Cells, Other
Species
Human, Mouse, Non-Human Primate, Other, Rat
Application
Flow Cytometry
Brand
ALDEFLUOR
Area of Interest
Cancer, Epithelial Cell Biology, Neuroscience, Stem Cell Biology
CAS Number
7647-01-0

Data Figures

Identification of ALDHbr cells from mouse embryonic brain samples

Figure 1. Identification of ALDHbr Cells from Mouse Embryonic Brain Samples

E14 SVZ cells stained with ALDEFLUOR™. FACS profiles of DEAB control (A) and ALDH staining (B).

Identification of ALDHbr SSC LO cells from human hematopoietic samples

Figure 2. Identification of ALDHbr SSC LO Cells from Human Hematopoietic Samples

Bone marrow low density cells (A-B), peripheral blood mononuclear cells (C, D) and umbilical cord blood cells (E, F) stained with ALDEFLUOR™. FACS profiles of DEAB control (A, C, E) and ALDH staining (B, D, F).

Identification of ALDHbr cells from human breast cancer cell lines

Figure 3. Identification of ALDHbr Cells from Human Breast Cancer Cell Lines

SKBR3 breast cancer cells stained with ALDEFLUOR™ for 45 minutes. FACS profiles of DEAB control (A) and ALDH staining (B).

Identification of ALDHbr Cells from human mammary epithelial samples

Figure 4. Identification of ALDHbr Cells from Human Mammary Epithelial Samples

Primary normal human mammary epithelial samples stained with ALDEFLUOR™. FACS profiles of DEAB control (A) and ALDH staining (B).

Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Document Type
Product Name
Catalog #
Lot #
Language
Document Type
Product Name
Catalog #
01700
Lot #
Lot #1000126956 or higher for Catalog # 01700
Language
English
Document Type
Product Name
Catalog #
01700
Lot #
All
Language
English
Document Type
Product Name
Catalog #
01700
Lot #
All
Language
English
Document Type
Product Name
Catalog #
01700
Lot #
All
Language
English
Document Type
Product Name
Catalog #
01700
Lot #
All
Language
English
Document Type
Product Name
Catalog #
01700
Lot #
All
Language
English

Applications

Resources and Publications

Educational Materials (23)

Brochure
Brochure
Brochure
Brochure
Brochure
Brochure
Identification of Viable Stem and Progenitor Cells with ALDEFLUORâ„¢
Technical Bulletin
Identification of Viable Stem and Progenitor Cells with ALDEFLUORâ„¢
ALDEFLUORâ„¢ Assay Optimization
Technical Bulletin
ALDEFLUORâ„¢ Assay Optimization
Identification of ALDH-Expressing Cancer Stem Cells Using ALDEFLUORâ„¢
Technical Bulletin
Identification of ALDH-Expressing Cancer Stem Cells Using ALDEFLUORâ„¢
The Basic FACS on ALDEFLUORâ„¢: The Quick Guide to Flow Cytometry
Technical Bulletin
The Basic FACS on ALDEFLUORâ„¢: The Quick Guide to Flow Cytometry
How to Identify ALDH-Expressing Cells in Human Breast Tissue
Protocol
How to Identify ALDH-Expressing Cells in Human Breast Tissue
SnapShot: Breast Cancer
Wallchart
SnapShot: Breast Cancer
The Identity and Properties of Mesenchymal Stem Cells
Wallchart
The Identity and Properties of Mesenchymal Stem Cells
How to Detect and Isolate Normal and Cancer Stem Cells Based on ALDH Activity with ALDEFLUORâ„¢
1:22
Video
How to Detect and Isolate Normal and Cancer Stem Cells Based on ALDH Activity with ALDEFLUORâ„¢
Flow Cytometry Analysis of ALDH Bright Cells with the ALDEFLUORâ„¢ Assay Kit
6:29
Video
Flow Cytometry Analysis of ALDH Bright Cells with the ALDEFLUORâ„¢ Assay Kit
Development and Validation of a Rapid ALDHbr-Based Cord Blood Potency Assay
41:09:00
Video
Development and Validation of a Rapid ALDHbr-Based Cord Blood Potency Assay
Optimization of ALDEFLUORâ„¢ Protocol
4:44
Video
Optimization of ALDEFLUORâ„¢ Protocol
ALDH+ Cancer Precursor Cells in Drug Response Prediction
42:24:00
Video
ALDH+ Cancer Precursor Cells in Drug Response Prediction
º£½ÇÆƽâ°æ Journal Club: Mechanisms of Glioblastoma Resistance
29:15
Video
º£½ÇÆƽâ°æ Journal Club: Mechanisms of Glioblastoma Resistance
Bright Ideas Podcast: ALDH in Breast Cancer Treatment Response
11:03
Video
Bright Ideas Podcast: ALDH in Breast Cancer Treatment Response
Tools for Breast Cancer Research
1:23
Video
Tools for Breast Cancer Research
ALDEFLUORâ„¢ and Cancer Stem Cells
47:13:00
Video
ALDEFLUORâ„¢ and Cancer Stem Cells
Potential Applications of ALDH Bright Cells In Regenerative Medicine
53:18
Webinar
Potential Applications of ALDH Bright Cells In Regenerative Medicine

Frequently Asked Questions

The reagents in the kit were frozen when I received it. Will this cause a problem?

No, the reagents in the kit are stable to freezing. Assay performance will not be affected.

Is it acceptable for activation of the ALDEFLUOR™ reagent to exceed 30 minutes?

Yes, as long as room temperature does not exceed 22°C, the reaction can proceed for up to 6 hours with no effect on the assay.

Can I speed up the activation reaction by incubating at 37°C?

This is not recommended. Incubation of the activation reaction at 37°C will not significantly speed up the reaction, and degradation of the activated substrate will occur more quickly at higher temperatures.

Will the activation reaction proceed at refrigerator (2 - 8°C) temperatures?

The ALDEFLUOR™ reagent will remain active for 1 week when stored at 2 - 8°C. For longer storage, divide the remaining reagent into aliquots and store at or below -20°C. Activated ALDEFLUOR™ reagent is stable for 1 year when stored frozen.

How should I store the ALDEFLUOR™ reagent after it is activated?

The ALDEFLUOR™ reagent will remain active for 1 week when stored at 2 - 8°C. For longer storage, divide the remaining reagent into aliquots and store at or below -20°C. Activated ALDEFLUOR™ reagent is stable for 1 year when stored frozen.

Why must the ALDEFLUOR™ assay buffer be added?

This assay has been optimized for detecting stem and progenitor cells by addition of the ALDEFLUOR™ assay buffer. Stem and progenitor cells have high ABC transporter activity and BAAA is a substrate for these efflux pumps. The assay buffer incorporates an efflux pump inhibitor to produce optimal discrimination of the ALDHbr cells and to maximize fluorescent signal stability. We thus recommend that cells be kept on ice and that the ALDEFLUOR™ assay buffer be used throughout all procedures performed after ALDH staining. Not using the assay buffer produces a proportionate loss in the assay signal, depending on the time and temperature at which the stained cells are held.

Is it acceptable for the staining reaction to exceed 30 minutes?

It depends on the cell type. With hematopoietic cells the reaction time can be up to 1 hour at 37°C with no effect on the fluorescence intensity. Incubation periods exceeding 1 hour may lead to an weaker signal and/or higher background. For nonhematopoietic cells optimal incubation times may be different. For example, for the human mammary epithelial SKBR3 cell line, the optimal incubation time was 45 minutes in experiments done at º£½ÇÆƽâ°æ. It is recommended to test different incubation times and determine the optimal incubation time for different cell types.

Will the staining reaction proceed at refrigerator (2 - 8°C) temperatures?

Yes, but full staining will take at least 3 - 4 hours. The staining reaction can continue for up to 24 hours at 2 - 8°C without any effect on the assay.

Can I add any other efflux inhibitors to the ALDEFLUOR™ assay buffer?

Yes. To prevent efflux of the activated ALDEFLUOR™ reagent and the reaction product, the following may be added individually or in combination. These reagents may also improve discrimination of the ALDHbr population, but results will vary by sample type.
• 50 - 100 µM verapamil
• 2.5 mM probenecid
• 100 mM 2-deoxy-D-glucose
• 1 mg/mL sodium azide (0.1%) Note: Sodium azide may be toxic to cells. Do not use if cellular function assays are to be performed after the ALDEFLUOR™ assay.
Note: Ice is the universal efflux inhibitor. Keep all ALDEFLUOR™-reacted samples on ice or at 2 - 8°C as much as possible.

Can I stain the cells at a concentration higher than 1 x 106 cells/mL?

Increasing the concentration of cells up to 5-fold the recommended concentration should have no effect on performance of the assay when using human blood cells. Increasing cell concentrations greater than 5-fold the recommended concentration will decrease assay signal and thereby decrease discrimination of the ALDHbr population. However, different cell types may produce different results. Cell titration experiments may be necessary to determine the optimal cell concentration for different cell types. To stain large number of cells it may be better to increase the sample and reagent volume.

What anticoagulants can be used to collect samples?

Optimal assay performance can be achieved with peripheral blood and leukapheresis samples anticoagulated with acid-citrate dextrose (ACD), ethylenediaminetetraacetic acid (EDTA), or sodium heparin. Bone marrow should be anticoagulated with sodium heparin. Cord blood units may be collected into citrate phosphate dextrose anticoagulant.

Do erythrocytes (red blood cells) interfere with the assay?

The large number of erythrocytes present in peripheral blood, apheresis collections, bone marrow, and umbilical cord blood samples can compete with stem/progenitor cells for the ALDEFLUOR™ substrate. For optimal assay performance, lyse the erythrocytes by treating the samples with ammonium chloride. The ratio of lysis buffer to cell numbers or blood volume must be optimized (10 to 40 parts buffer to sample), and the time (10 - 30 minutes) and temperature (RT or 2 - 8°C) of incubation must be carefully controlled for each lysis buffer and sample type.

What solutions can be used to lyse erythrocytes?

Optimal erythrocyte lysis can be achieved with buffers containing:
• Ammonium chloride (e.g. 0.17 M NH4CI, 10 mM Tris HCI, 0.25 mM EDTA),
• 1X ABC Lysis Buffer (eBioscience, San Diego, CA)
• VitaLyse® (BioE, St Paul, MN).
We do not recommend use of the following or any other solution that contains a fixative, as these will render the cells nonviable:
• CyLyse® (Partec GMBH, Munster, Germany),
• FACS™ Lysing solution (BD Biosciences, San Jose, CA.)

Can fixed cells be used with this assay?

No. The ALDEFLUOR™ reagent is a substrate for the enzyme aldehyde dehydrogenase. ALDEFLUOR™ is a viability marker since the substrate is taken up, catalyzed and retained only by viable cells. It is important to ensure that reagents used for erythrocyte lysis do not contain a fixative.

Does the ALDEFLUOR™ assay work on cryopreserved cells?

ALDEFLUOR™ has been extensively tested on fresh and cryopreserved umbilical cord blood, peripheral blood and leukapheresis samples from patients and mobilized donors. If done correctly, cryopreservation and thawing should not cause loss in cell viability or fluorescence intensity of ALDHbr cells. As only viable cells retain the ALDEFLUOR™ reaction product, a loss in viability will be reflected as a decrease in the percentage of ALDHbr cells and an increase in the percentage of dead/dying cells (detectable by staining for propidium iodide or other viability dyes).

Will ALDEFLUOR™ buffer prevent efflux in cells from non-hematopoietic tissues or from other species?

The proprietary ALDEFLUOR™ assay buffer has been designed to optimize the detection of ALDH-positive (or ALDHbr) cells in human blood. The buffer contains an ATP-binding cassette (ABC) transport inhibitor that prevents active efflux of the ALDEFLUOR™ product from these cells. This transport inhibitor may not prevent efflux from other tissue types or from other species. Consequently, when using samples other than human blood, following the incubation with the activated ALDEFLUOR™ reagent at 37°C, the reacted cells should be kept at 2 - 8°C to prevent efflux, and thus the loss of fluorescence. For a list of additional efflux inhibitors that may be added to the ALDEFLUOR™ buffer see the "CAN I ADD ANY OTHER EFFLUX INHIBITORS TO THE ALDEFLUOR™ ASSAY BUFFER?" question.

Will DEAB inhibit ALDH activity in cells from non-hematopoietic tissues or from other species?

The specific ALDH gene product expressed in non-human, non-blood products may not be inhibited by DEAB. A lack of difference between test and negative control samples may indicate that the inhibitor was not effective, or that there is no ALDH activity in the cells in the sample. Kinetic studies (a progressive increase in ALDEFLUOR™ fluorescence in the negative control tube with time of reaction) may be useful to differentiate these two alternatives. Other ALDH inhibitors can be used as appropriate for the enzyme isoform expressed. For example, Disulfuram inhibits several mammalian ALDH gene products.

Can I use a greater concentration of the ALDEFLUOR™ substrate to improve the discrimination of the ALDHbr population?

When staining non-blood products, it may be necessary to titrate the ALDEFLUOR™ substrate to determine the optimal concentration. We suggest a range of concentrations from 5-fold less to 10-fold more than the standard concentration. During titration we recommend maintaining the concentration of DEAB at 10-fold molar excess of activated ALDEFLUOR™ reagent, and therefore, it is necessary to adjust the amount of DEAB when titrating the substrate.

Can I analyze cells by the ALDEFLUOR™ assay and the side population assay simultaneously?

Yes, the side population assay can be performed in conjunction with the ALDEFLUOR™ assay (Pearce and Bonnet. Exp Hematol 35: 1437-1446, 2007). The Side Population assay should be performed first, followed by the ALDEFLUOR™ assay. We recommend adding 50 µM verapamil to the ALDEFLUOR™ assay buffer when performing both assays.

Why are all the cells in the cytogram fluorescent to some degree?

The ALDEFLUOR™ substrate is a non-polar fluorescent molecule that freely diffuses into all cells. In the DEAB-treated control, fluorescence will reflect the size of the intracellular substrate pool. Fluorescence in the test sample will additionally reflect ALDH activity. Human stem and progenitor cells typically have more ALDH activity than mature cells, and this quantitative difference allows stem cells to be resolved from the other cells.

How do I compensate for multiparameter flow analysis when the staining of ALDHbr cells is so bright?

We would recommend washing your cells with ALDEFLUOR™ assay buffer after the reagent reaction to eliminate background fluorescence from excess substrate. The ALDEFLUOR™ reagent shows an emission spectrum similar to FITC with peak emission at 512 nm. Due to spectral overlap of the ALDEFLUOR™ reagent with fluorochromes that are detected below 650 nm, we recommend using antibodies conjugated to fluorochromes that emit at higher wavelengths for antigens which typically exhibit low levels of expression. For example, when studying the coexpression of CD34 on ALDHbr cells we used the antibody combination, CD45 phycoerythrin (PE), 7- aminoactinomycin D (7-AAD) and CD34 allophycocyanin (APC). Due to the brightness of the ALDEFLUOR™ reagent fluorophore, we strongly recommend the use of compensation controls for every experiment. Adequate compensation will not be achieved with commercially available fluorescent beads.

Publications (324)

C1orf226 Promotes Glioma Stemness and Oncogenesis in Glioma via PLK1-Mediated Wnt/β-Catenin Signaling. Y. Lu et al. Cancer medicine 2026 Jun

Abstract

INTRODUCTION: Glioma stem cells (GSCs) are pivotal drivers of tumor progression and therapeutic resistance; however, the underlying regulatory mechanisms have not been fully elucidated. This study aimed to identify novel oncogenic drivers in glioma via a comprehensive multiomics analysis. METHODS: We integrated three datasets to screen potential oncogenic drivers. Functional experiments were performed to examine the effects of C1orf226 on glioma cell proliferation, cell cycle, epithelial-mesenchymal transition (EMT), and apoptosis. Additionally, tumor sphere formation assays, ALDH+/CD133+ cell detection, and stemness marker analysis were used to evaluate the role of C1orf226 in GSC stemness. Mechanistic studies involving immunoprecipitation (IP) and ubiquitination assays were applied to characterize the interaction between C1orf226 and PLK1, as well as their regulation on β-catenin stability and Wnt/β-catenin signaling. Rescue assays further verified the functional crosstalk between C1orf226 and PLK1. Xenograft mouse models were used in vivo to assess the impacts of C1orf226 knockdown on tumor growth and stemness markers. RESULTS: C1orf226 was significantly upregulated in glioma tissues compared with normal brain tissues. Its high expression correlated with shorter patient survival and served as an independent prognostic factor in the CGGA cohort. In vitro, C1orf226 exerted oncogenic function by facilitating cell proliferation, cell cycle progression, EMT-like processes, and stemness, and suppressing apoptosis. Mechanistically, C1orf226 interacts with PLK1 to block its degradation, thereby activating PLK1 and downstream Wnt/β-catenin signaling. Rescue experiments verified that PLK1 overexpression restored β-catenin stability, Wnt/β-catenin target gene expression, and GSC properties impaired by C1orf226 knockdown. In vivo, C1orf226 knockdown restrained xenograft growth and reduced stemness marker levels, and PLK1 co-expression abrogated these effects. CONCLUSIONS: Our findings reveal that C1orf226 is a PLK1-dependent regulator of Wnt/β-catenin signaling and GSC plasticity, highlighting its potential as a promising therapeutic target in glioma.
The PCNA inhibitor AOH1996 suppresses cancer stemness and enhances anti-PD1 immunotherapy in squamous cell carcinoma Y. Wang et al. Stem Cell Research & Therapy 2025 Sep

Abstract

Proliferating cell nuclear antigen (PCNA), a well-documented anticancer target, is critical for DNA synthesis, replication, and repair. AOH1996, a small-molecule PCNA inhibitor, is currently undergoing clinical trials for the treatment of advanced solid tumors. However, the therapeutic effect of AOH1996 on head and neck squamous cell carcinoma (HNSCC) remains unclear. The effects of AOH1996 on HNSCC biological behaviors and cancer stemness were tested in HNSCC cells and nude mice. The combination treatment of AOH1996 and anti-PD1 was performed in a 4-nitroquinoline N-oxide (4NQO)-induced HNSCC mouse model. RNA sequencing, Western Blotting, immunofluorescence staining, comet assays, and qRT‒PCR were conducted for mechanistic studies. Our results showed that AOH1996 effectively inhibited HNSCC proliferation and invasion both in vitro and in vivo. AOH1996 suppressed HNSCC stemness, development, and metastasis. Moreover, AOH1996 altered the tumor immune microenvironment into an inflamed state with increased CD8 + T-cell infiltration, rendering it a favorable partner for combination therapy with immune checkpoint inhibitors. Mechanistically, AOH1996 induced cellular DNA damage, suppressed cancer stemness through the upregulation of p-TBK1, and promoted the secretion of CD8 + T-cell-recruiting chemokines by stimulating IRF3-mediated transcription. Taken together, our results demonstrated that AOH1996 suppressed tumor growth, eliminated cancer stem cells (CSCs), and synergistically enhanced the efficacy of anti-PD1 immunotherapy in HNSCC. The online version contains supplementary material available at 10.1186/s13287-025-04607-9.
Chromatin looping-based CRISPR screen identifies TLK2 as chromatin loop formation regulator in cancer stemness plasticity Z. Wang et al. Nature Communications 2025 Oct

Abstract

Targeting cancer cell plasticity through chromatin organization is an emerging research area, yet the molecular mechanisms that govern chromatin loop formation remain unclear. Here, we develop a CRISPR screen based on our engineered live-cell CTCF-cohesin contact reporters to identify regulators of chromatin loops. Our findings reveal that tousled-like kinase 2 (TLK2) functions as a key regulator of chromatin loop formation during the cancer stemness transition. Mechanistically, TLK2 phosphorylates DYNLL1, enhancing its interaction with CTCF to promote CTCF-cohesin hub formation at the KLF4 locus. Suppressing TLK2 impairs cancer stemness plasticity, sensitizes cancer cells to cytotoxic stress in vitro, and reduces lung metastases and enhances immunotherapy response in breast cancer mouse models. Clinically, elevated TLK2 expression correlates with poor prognosis in breast cancer patients. Collectively, these findings identify TLK2 as a potential therapeutic target for mitigating cancer stemness plasticity, highlighting chromatin loop-targeting therapy as a promising strategy to eradicate cancer stem cells. Aberration of chromatin organisation is linked to cancer progression and cancer cell plasticity. Here, the authors investigate changes in chromatin state during the transition of cancer cells to cancer stem cells and, using a CTCF-cohesin contact reporter CRISPR screen, identify TLK2 as a driver of plasticity via regulation of chromatin loop formation.
View All Publications

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