ALDECOUNT® IVD Assay Kit with Lysis Buffer

Procedures and instruction manuals:

•  PRODUCT INFORMATION SHEET - 01720, Lot# All:29889-PIS_1_0_0.pdf

Educational resources:

•  VIDEO: ALDEFLUOR™: Detect Normal and Cancer Precursor Cells
•  WEBINAR: ALDEFLUOR™ and Cancer Stem Cells
•  TECH BULLETIN: Immunophenotyping of ALDEFLUOR™-Reacted Cells
•  TECH BULLETIN: Identification of viable stem and progenitor cells with ALDEFLUOR™ (Catalog #28728)
•  TECH BULLETIN: A guide to identification of stem cells without antibodies (Catalog #29140)
•  BROCHURE: The Basic FACS of ALDEFLUOR™: The Quick Guide to Flow Cytometry
•  VIDEO: The Basic FACS About ALDEFLUOR™ - A Guide To Successful Flow Cytometry Analysis
•  BROCHURE: Tips for Optimizing ALDEFLUOR™ Assay Conditions
•  VIDEO: Bright Ideas Podcast: ALDH in Breast Cancer Treatment Response

MSDS:

•  01720-MSDS.pdf
•  01721-MSDS.pdf

FAQS:

• 
  ALDEFLUOR™ PROCEDURE: REAGENT ACTIVATION AND STORAGE
  
  
  • 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. IS IT ACCEPTABLE FOR ACTIVATION OF THE ALDEFLUOR™ REAGENT TO EXCEED 30 MINUTES?
    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 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.
  
  ALDEFLUOR™ PROCEDURE: STAINING REACTION
  
  
  • Q. 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.
  • Q. IS IT ACCEPTABLE FOR THE STAINING REACTION TO EXCEED 30 MINUTES?
    A. 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 STEMCELL. It is recommended to test different incubation times and determine the optimal incubation time for different cell types.
  • 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. 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 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.
  • Q. 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. 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.
  
  DETECTING HEMATOPOIETIC CELLS
  
  
  • 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. 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.
  • 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 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. DOES THE ALDEFLUOR™ ASSAY WORK ON CRYOPRESERVED CELLS?
    A. 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).
  
  DETECTING NON-HEMATOPOIETIC CELLS
  
  
  • 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.
  
  TISSUE-SPECIFIC PROTOCOL OPTIMIZATION
  
  
  • 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.
  
  ALDEFLUOR™-COMPATIBLE ASSAYS
  
  
  • Q. CAN I ANALYZE CELLS BY THE ALDEFLUOR™ ASSAY AND THE SIDE POPULATION ASSAY SIMULTANEOUSLY?
    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 adding 50 µM verapamil to the ALDEFLUOR™ assay buffer when performing both assays.
  • Q. WHY ARE ALL 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. 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.
  • Q. HOW DO I COMPENSATE FOR MULTIPARAMETER FLOW ANALYSIS WHEN THE STAINING OF ALDHBR CELLS IS SO BRIGHT?
    A. 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.

This product has been used in:

1. Olivier Pierre-Louis et al. Dual SP/ALDH Functionalities Refine The Human Hematopoietic Lin(-) CD34(+) CD38(-) Stem/Progenitor Cell Compartment.Stem Cells 27 (10) 2552-2562 (July 30, 2009)
2. Thomas J Povsic et al. Circulating progenitor cells can be reliably identified on the basis of aldehyde dehydrogenase activity.J Am Coll Cardiol 50 (23) 2243-2248 (December 4, 2007)
3. James Kurtz et al. Assessment of cord blood hematopoietic cell parameters before and after cryopreservation.Transfusion 47 (9) 1578-1587 (September 2007)
4. Oliver Christ et al. Improved purification of hematopoietic stem cells based on their elevated aldehyde dehydrogenase activity.Haematologica 92 (9) 1165-1172 (August 1, 2007)
5. M V Lioznov et al. Aldehyde dehydrogenase activity as a marker for the quality of hematopoietic stem cell transplants.Bone Marrow Transplant 35 (9) 909-914 (May 2005)
6. Maxim A Vodyanik et al. Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential.Blood 105 (2) 617-626 (January 15, 2005)
7. David A Hess et al. Functional characterization of highly purified human hematopoietic repopulating cells isolated according to aldehyde dehydrogenase activity.Blood 104 (6) 1648-1655 (September 15, 2004)
8. R J Jones et al. Assessment of aldehyde dehydrogenase in viable cells.Blood 85 (10) 2742-2746 (May 15, 1995)