ƽ

  • Compare Products
Menu
Account
Access free e-books featuring techniques, tips, and protocols to move your research forward.   Explore Now >

ALDEFLUOR™ Assay Buffer

Additional assay buffer for use with the ALDEFLUOR™ kit

Loading...
 
 
New look, same high quality and support! You may notice that your instrument or reagent packaging looks slightly different from images displayed on the website, or from previous orders. We are updating our look but rest assured, the products themselves and how you should use them have not changed. Learn more

ALDEFLUOR™ Assay Buffer

Additional assay buffer for use with the ALDEFLUOR™ kit

Catalog #
(Select a product)
Additional assay buffer for use with the ALDEFLUOR™ kit
Request Pricing Request Pricing

What's Included

  • ALDEFLUOR™ Assay Buffer (Catalog #01702)
    • ALDEFLUOR™ Assay Buffer, 55 mL
Products for Your Protocol
To see all required products for your protocol, please consult the Protocols and Documentation.
  1. ALDEFLUOR™ Kit
    ALDEFLUOR™ Kit

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

Overview

Use ALDEFLUOR™ Assay Buffer to optimize the detection of aldehyde dehydrogenase-bright (ALDHbr) cells and maximize fluorescent signal stability during the ALDEFLUOR™ assay.

Available either individually or as part of the ALDEFLUOR™ Kit, ALDEFLUOR™ Assay Buffer is recommended for use in all procedures performed after ALDH staining to inhibit the active efflux of the ALDEFLUOR™ reaction product and to avoid loss in the assay signal.

For detailed information on protocols for using the ALDEFLUOR™ Assay Buffer, please refer to the Product Information Sheet (PIS).
Species
Human, Mouse, Non-Human Primate, Other, Rat
Brand
ALDEFLUOR
Area of Interest
Cancer, Epithelial Cell Biology, Neuroscience, Stem Cell Biology

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 #
01702
Lot #
Lot #1000126956 or higher for Catalog # 01700
Language
English
Document Type
Product Name
Catalog #
01702
Lot #
All
Language
English

Applications

Resources and Publications

Educational Materials (22)

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

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 ƽ. 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 (285)

Combined CHK1 and PD-L1 blockade as a novel therapeutic strategy against stemness and immunosuppression in ovarian cancer M. Chen et al. Cancer Immunology, Immunotherapy : CII 2025 Nov

Abstract

BackgroundCancer stem cells (CSCs) are considered the ‘seeds’ of recurrence after chemotherapy, but eliminating CSCs remains notoriously challenging. This study aims to examine whether cell cycle checkpoint kinase 1 (CHK1) blockade can abrogate the stemness of ovarian cancer (OC) cells, making them easier targets of anti-tumor immunity. Methods: Prexasertib was used to block CHK1 in OC cell lines and xenografts, and its cytotoxicity was assessed in vitro and in vivo. In vitro tumor-sphere formation assays and stemness markers were used to evaluate cell stemness. PD-L1 expressions were examined via qRT-PCR, Western blot, flow cytometry, and immunohistochemistry. Prexasertib in combination with anti-PD-L1 antibody Atezolizumab was tested in immune-proficient mice bearing OC xenografts in terms of effects on tumor growth, tumor cell stemness, and tumor infiltrating lymphocytes via tumor volume monitoring, immunohistochemistry, and flow cytometry. Results: Prexasertib effectively inhibited CHK1 phosphorylation, exhibited significant anti-tumor effects in vitro and in vivo, accompanied by decreased OC cell stemness. CHK1 was highly expressed in tumor spheres versus tumor cells cultured in 2D system, and Prexasertib treatment suppressed sphere formation and reduced the ALDH+ cell fraction. Unexpectedly, Prexasertib upregulated PD-L1 expression in tumor cells. In vivo, combining Prexasertib with Atezolizumab led to more remarkable remission of tumors, when compared with Prexasertib or Atezolizumab alone. Meanwhile, the tumor-infiltrating CD8+ T cells significantly increased in the combination group, while exhausted T cells decreased; the treatments did not affect CD4+ cell infiltration. Conclusions: Dual targeting of CHK1 and PD-L1 may improve OC treatment by simultaneously suppressing stemness and enhancing anti-tumor immunity.
mTORC1‐USP30‐LEF1 Cascade Regulates Cancer Stemness and Malignant Progression Through Mitonuclear Crosstalk X. Li et al. MedComm 2025 Nov

Abstract

USP30, a ubiquitin‐specific protease, primarily characterized as a mitochondrial deubiquitinase regulating mitophagy, has not been previously reported to have nuclear functions. In this study, we demonstrate that USP30 is present in both mitochondrial and nuclear compartments. Nutrient deprivation triggers USP30 nuclear translocation via an N‐terminal nuclear localization signal (NLS), mediated through suppression of mTORC1‐dependent phosphorylation at serine 104, a modification constraining nuclear entry. Nuclear USP30 acts as a tumor suppressor by inhibiting cancer stemness and chemoresistance in triple‐negative breast cancer (TNBC) cells. Mechanistically, USP30 directly interacts with and deubiquitinates the transcription factor TCF/LEF1 at K379 and K382 residues, disrupting recruitment of CBP/P300 co‐activators to the β‐catenin/LEF1 complex. This abolishes β‐catenin/LEF1 transactivation and suppresses WNT signaling. Clinically, USP30 is downregulated in TNBC and cancer stem cells (CSCs), with notably reduced nuclear levels in cancer tissues. Overexpression of nuclear USP30 markedly reduces lung metastatic burden in TNBC mouse models. These findings uncover a novel role for nuclear USP30 in regulating cancer stemness and suggest that targeting the dynamic relocalization of USP30 from mitochondria to the nucleus could offer new therapeutic strategies for breast cancer metastasis.
CXCR4 + mammary gland macrophageal niche promotes tumor initiating cell activity and immune suppression during tumorigenesis E. Lee et al. Nature Communications 2025 May

Abstract

Tumor-initiating cells (TICs) share features and regulatory pathways with normal stem cells, yet how the stem cell niche contributes to tumorigenesis remains unclear. Here, we identify CXCR4 + macrophages as a niche population enriched in normal mammary ducts, where they promote the regenerative activity of basal cells in response to luminal cell-derived CXCL12. CXCL12 triggers AKT-mediated stabilization of β-catenin, which induces Wnt ligands and pro-migratory genes, enabling intraductal macrophage infiltration and supporting regenerative activity of basal cells. Notably, these same CXCR4 + niche macrophages regulate the tumor-initiating activity of various breast cancer subtypes by enhancing TIC survival and tumor-forming capacity, while promoting early immune evasion through regulatory T cell induction. Furthermore, a CXCR4 + niche macrophage gene signature correlates with poor prognosis in human breast cancer. These findings highlight the pivotal role of the CXCL12-CXCR4 axis in orchestrating interactions between niche macrophages, mammary epithelial cells, and immune cells, thereby establishing a supportive niche for both normal tissue regeneration and mammary tumor initiation. Subject terms: Cancer stem cells, Cancer microenvironment, Tumour immunology
View All Publications

Related Products

Related Products
New look, same high quality and support! You may notice that your instrument or reagent packaging looks slightly different from images displayed on the website, or from previous orders. We are updating our look but rest assured, the products themselves and how you should use them have not changed. Learn more
Legal Statement:

ALDEFLUOR is registered trademark of ALDAGEN Inc.

Quality Statement:

PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED. FOR ADDITIONAL INFORMATION ON QUALITY AT ƽ, REFER TO WWW.ƽ.COM/COMPLIANCE.

Copyright © 2025 ƽ. All rights reserved.