ALDECOUNT® IVD Assay Kit with Lysis Buffer

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ALDECOUNT® Assay Kit

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  • ALDECOUNT Box
ALDECOUNT Box
ALDECOUNT® is used to identify and enumerate by flow cytometry cells that express high levels of aldehyde dehydrogenase (ALDHbr) in human peripheral blood, leukapheresis, bone marrow and umbilical cord blood samples. The test is for in vitro diagnostic use and is suited only for professional use in specialized clinical laboratories.
Product Name Description Catalog # Size Price Quantity
ALDECOUNT® IVD Assay Kit with Lysis Buffer To enumerate ALDH-bright cells in fresh and frozen clinical samples. for general diagnostic purposes. (Available in USA only) 01720 1 Kit 505.00 USD      
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ALDECOUNT® RUO Assay Kit with Lysis Buffer To enumerate ALDH-bright cells in fresh and frozen clinical samples. for general diagnostic purposes. (RUO only, available worldwide) 01721 1 Kit Request pricing      

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Recommended for:
This device is to be used for general diagnostic purposes to enumerate ALDHbr cells in fresh or frozen clinical samples
 
These samples can be from donors mobilized with granulocyte colony stimulating factor (G-CSF) or some other mobilizing agent, or from patients that have been mobilized or treated with therapeutic agents
 
This device is to be used by highly trained laboratory personnel skilled in cell staining and flow cytometric analysis
Components:
• ALDECOUNT® reagent tubes, 20 units
• Control tubes, 20 units
• Activator, 1 vial
• DEAB Reagent in ethanol, 1 vial
• Neutralization buffer, 1 bottle
• ALDECOUNT® Assay buffer, 1 bottle
Intended Use Statement: For in vitro diagnostic use only
Equipment Required:
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Legal Statement: ALDECOUNT® is a registered trademark of ALDAGEN Inc.
Product Type: Stem cell detection kits
Application: In vitro diagnostic
Area of Interest: Cord blood banking, Hematopoietic stem cell research, Stem cell biology
Cell Type: Hematopoietic stem & progenitor cells
Popular Product Line: ALDECOUNT, ALDEFLUOR

Procedures and instruction manuals:

Educational resources:

MSDS:

FAQS:


  • Q. FOR WHAT SAMPLE SOURCES HAS THE ALDEFLUOR® ASSAY BEEN OPTIMIZED?

    A. The assay has been optimized for use with human peripheral blood and leukapheresis products. These samples can be obtained from normal donors, donors 'mobilized' by treatment with any number of cytokines and growth factors, and patients whose stem cells have been mobilized by chemotherapeutic or other agents. The ALDEFLUOR® assay is also compatible with bone marrow and umbilical cord blood samples.

  • Q. WHICH ALDEHYDE DEHYDROGENASE ENZYME IS ALDEFLUOR® OPTIMIZED FOR?

    A. Seventeen isozymes belong to the human aldehyde dehydrogenase superfamily (Sládek NE. J Biochem Mol Toxicol 17: 7-23, 2003). These enzymes vary in tissue distribution and preferred substrate. The ALDEFLUOR® kit is optimized for interaction with human ALDH 1A1. Its performance with other enzymes within the human superfamily or with other species has not been determined. While most major tissues expres some form of ALDH, the ALDH expressed may not be able to oxidize the ALDEFLUOR® substrate.

  • Q. CAN THE ALDEFLUOR® REAGENT BE USED TO IDENTIFY STEM CELLS FROM NON-HEMATOPOIETIC TISSUES OR FROM OTHER SPECIES?

    A. ALDEFLUOR® has been optimized for use with human cells; its performance with cells from other species has not been validated. We provide a protocol that has been optimized for the identification of ALDHbr cells from human blood products. However, it has been reported that ALDEFLUOR® can recognize:
    - Primitive hematopoietic progenitor cells in mouse bone marrow (Armstrong et al. Stem Cells 22: 1142-1151, 2004; Juopperi et al. Exp Hematol 35: 335-341, 2007)
    - Multipotent neural cells in embryonic rats (Cai et al. J Neurochem 88: 212-226, 2004)
    - Neural stem cells from mouse brain and spinal cord (Corti et al. Stem Cells 24: 975-985, 2006, Corti et al. Hum Mol Genet 15: 167-187, 2006, Louis et al., Society for Neuroscience 2005)
    - Endothelial and mesenchymal progenitor cells (Gentry et al. Cytotherapy 9: 259-274, 2007)
    - Normal and malignant mammary progenitors (Ginestier et al. Cell Stem Cell 1: 555-567, 2007)
    - Stem cells from the tunicate Botryllus (Laird et al. Cell 123: 1351-1360, 2005)
    Identification and isolation of ALDHbr cells from heterogeneous cell suspensions can be facilitated by enriching the sample for stem cells prior to performing the ALDEFLUOR® assay. We recommend enriching for stem cells by removing mature cells with a cocktail of antibodies to lineage-specific markers. A cocktail for enriching stem cells from mouse bone marrow is described by Armstrong et al. (Stem Cells 22: 1142-1151, 2004), and a cocktail for enriching umbilical cord blood stem cells is described by Hess et al. (Blood 104:1648-1655, 2004). STEMCELL Technologies Inc. provides enrichment cocktails, and if you contact us at techsupport@stemcell.com, we can suggest the appropriate cell separation kit based on your needs.
    The side scatter and green fluorescence profiles of ALDHbr cells from cell lines will be different than the profiles exhibited by ALDHbr cells of blood products. Often, the ALDHbr cells of cell lines will exhibit a higher and more heterogeneous side scatter profile, and the green fluorescence profile will be broader and perhaps less distinct when compared to the profiles exhibited by ALDHbr cells of blood products. When working with cell lines, it is helpful to use gating strategies that exclude dead cells by using a viability marker such as propidium iodide before gating on green fluorescence for ALDHbr cells. Additionally, it can be helpful to dilute the sample and mix it with a cell line known to have high ALDH activity (Moreb et al. Cytometry B Clin Cytom 72: 281-289, 2007, Storms et al. Proc Natl Acad Sci USA 96: 9118-9123, 1999).

  • Q. CAN I USE A GREATER CONCENTRATION OF THE ALDEFLUOR® SUBSTRATE TO IMPROVE THE DISCRIMINATION OF THE ALDHbr POPULATION?

    A. 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.

  • Q. WILL ALDEFLUOR® BUFFER PREVENT EFFLUX IN CELLS FROM NON-HEMATOPOIETIC TISSUES OR FROM OTHER SPECIES?

    A. 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.

  • Q. WILL DEAB INHIBIT ALDH ACTIVITY IN CELLS FROM NON-HEMATOPOIETIC TISSUES OR FROM OTHER SPECIES?

    A. 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.

  • Q. DOES THE ALDEFLUOR® ASSAY WORK ON CRYOPRESERVED CELLS?

    A. The ALDEFLUOR® assay has been extensively tested on fresh and cryopreserved umbilical cord blood, peripheral blood and leukapheresis samples from patients and mobilized donors. If cryopreservation and thawing are done correctly, there should be no loss in cell viability or fluorescence intensity of ALDHbr cells. Because 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, as detected by staining for propidium iodide or other viability dyes.

  • Q. WHAT ANTICOAGULANTS CAN BE USED TO COLLECT SAMPLES?

    A. 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.

  • Q. DO ERYTHROCYTES (RED BLOOD CELLS) INTERFERE WITH THE ASSAY?

    A. 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. Ammonium chloride treatment of the samples to lyse the erythrocytes, gives optimal assay performance. 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.

  • Q. WHAT SOLUTIONS CAN BE USED TO LYSE ERYTHROCYTES?

    A. 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 the 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.)

  • Q. CAN FIXED CELLS BE USED WITH THIS ASSAY?

    A. 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.

  • Q. THE REAGENTS IN THE KIT WERE FROZEN WHEN I RECEIVED IT. WILL THIS CAUSE A PROBLEM?

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

  • Q. WHAT IS THE MOLECULAR WEIGHT OF THE VARIOUS REAGENT FORMS?

    A. The molecular weight of BODIPY®-aminoacetaldehyde diethyl acetal (BAAA-DA) is 407.27; the molecular weight of BODIPY®-aminoacetaldehyde (BAAA) is 334.15; the molecular weight of BODIPY®-aminoacetate (BAA) is 349.14.

  • Q. IF ACTIVATION OF THE ALDEFLUOR® REAGENT EXCEEDS 30 MINUTES, IS THAT OK?

    A. 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

  • Q. CAN I SPEED UP THE ACTIVATION REACTION BY INCUBATING THE REAGENT AT 37°C?

    A. 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.

  • Q. WILL THE ACTIVATION REACTION PROCEED AT REFRIGERATOR (2 - 8°C) TEMPERATURES?

    A. 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.

  • Q. HOW SHOULD I STORE THE ALDEFLUOR® REAGENT AFTER IT IS ACTIVATED?

    A. 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.

  • Q. IF THE STAINING REACTION EXCEEDS 30 MINUTES IS THIS OK?

    A. The reaction can proceed for up to 1 hour at 37°C with no effect on the assay. Incubation periods exceeding 1 hour may lead to an increase in assay variation.

  • Q. WILL THE STAINING REACTION PROCEED AT REFRIGERATOR (2 - 8°C) TEMPERATURES?

    A. 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.

  • Q. WHY MUST THE ALDEFLUOR® ASSAY BUFFER BE USED FOR STAINING AND ALL SUBSEQUENT STEPS?

    A. Stem and progenitor cells have high ABC transporter activity and the ALDEFLUOR® reaction product is a substrate for these efflux pumps. The assay buffer has been formulated to produce optimal discrimination of the ALDHbr cells and to maximize stability of the signal by incorporation of an efflux pump inhibitor. We also recommend that cells be kept cold (on ice) and that the ALDEFLUOR® assay buffer be used throughout all procedures performed after ALDH staining, such as immunophenotyping, side population assay and/or cell sorting. If the ALDEFLUOR® assay buffer is not used, you can expect a proportionate loss in the assay signal, depending on time and temperature at which the stained cells are held.

  • Q. CAN I ADD ANY OTHER EFFLUX INHIBITORS TO THE ALDEFLUOR® ASSAY BUFFER?

    A. 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 uM 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.

  • Q. I RAN OUT OF THE ALDEFLUOR® ASSAY BUFFER BEFORE I RAN OUT OF THE ALDEFLUOR® REAGENT. CAN I PURCHASE MORE BUFFER?

    A. Yes. Additional bottles of buffer (Catalog #01701 and #01702) can be purchased from STEMCELL Technologies.

  • Q. I NEED TO STAIN A LARGE NUMBER OF CELLS. CAN I STAIN THE CELLS AT A CONCENTRATION HIGHER THAN 1 X 106 CELLS/ML?

    A. 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. However, different sample types may produce different results. Increasing cell concentrations greater than 5-fold the recommended concentration will decrease assay signal and thereby decrease discrimination of the ALDHbr population.

  • Q. CAN I ANALYZE CELLS BY THE ALDEFLUOR® ASSAY AND THE SIDE POPULATION ASSAY AT THE SAME TIME?

    A. 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 the addition of 50 uM verapamil to the ALDEFLUOR® assay buffer when performing both assays.

  • Q. WHY ARE ALL OF THE CELLS IN THE CYTOGRAM FLUORESCENT TO SOME DEGREE?

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

  • Q. I HAVE TROUBLE COMPENSATING FOR MULTIPARAMETER FLOW ANALYSIS BECAUSE THE STAINING OF ALDHbr CELLS IS SO BRIGHT. WHAT CAN I DO TO MAKE COMPENSATION EASIER?

    A. Following the ALDEFLUOR® reaction, we recommend washing the cells with ALDEFLUOR® assay buffer to eliminate background fluorescence due to excess substrate before performing multiparameter analysis. ALDEFLUOR® is compatible with staining for phenotypic analysis. 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. Technical Notes on immunophenotyping and compensation contain detailed protocols for setting up proper compensation controls and determining compensation settings.

Product Name

Description

Catalog #
ALDECOUNT® IVD Assay Kit with Lysis Buffer ALDECOUNT® Assay Kit 01720

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