No, the kit reagents are stable to freezing. Test performance is not affected.
Yes, as long as the room temperature does not exceed 22 °C, the reaction can last up to 6 hours without affecting the assay.
This is not recommended. Incubation of the activation reaction at 37 °C does not significantly accelerate the reaction and degradation of the activated substrate is faster at higher temperatures.
ALDEFLUOR™ Reagent remains active for 1 week when stored at 2-8°C. For longer storage, take an aliquot of the remaining reagent and store at -20°C or below. Activated ALDEFLUOR™ reagent has a 1-year shelf life when stored frozen.
ALDEFLUOR™ Reagent remains active for 1 week when stored at 2-8°C. For longer storage, take an aliquot of the remaining reagent and store at -20°C or below. Activated ALDEFLUOR™ reagent has a 1-year shelf life when stored frozen.
This assay has been optimized for the detection of stem and progenitor cells by the addition of ALDEFLUOR™ Assay Buffer. Stem and progenitor cells have high ABC transporter activity, and BAAA is a substrate for these efflux pumps. The assay buffer contains an efflux pump inhibitor for optimal discrimination of ALDHbr cells and to maximize fluorescence signal stability. Therefore, we recommend storing cells on ice and using ALDEFLUOR™ Assay Buffer for any procedures performed after ALDH staining. If the assay buffer is not used, there will be a proportional loss of assay signal as a function of time and temperature at which the stained cells are maintained.
It depends on the type of cell. For hematopoietic cells, the reaction time can be up to 1 hour at 37°C without affecting the fluorescence intensity. Incubation times greater than 1 hour may result in a lower signal and/or higher background. For non-hematopoietic cells, the optimal incubation times may be different. For example, the optimal incubation time for the SKBR3 human mammary epithelial cell line was 45 minutes in experiments performed in STEMCELL. It is recommended to try different incubation times and determine the optimal incubation time for different cell types.
Yes, but the complete coloring takes at least 3-4 hours. The staining reaction can be continued for up to 24 hours at 2-8°C without affecting the assay.
Yes. To prevent leakage of the activated ALDEFLUOR™ reagent and reaction product, the following may be added individually or in combination. These items can also improve discrimination of the ALDHbr population, but the results vary depending on the type of sample.
• 50 - 100 µM verapamil
• Probenecid 2,5 mM
• 2-Deoxy-D-Glucose 100 mM
• 1 mg/ml sodium azide (0.1%) Note: sodium azide may be toxic to cells. Do not use if cell function assays are to be performed after the ALDEFLUOR™ assay.
Note: Ice is the universal efflux inhibitor. If possible, store all samples that reacted with ALDEFLUOR™ on ice or at 2-8°C.
Increasing the cell concentration up to 5 times the recommended concentration should not affect test performance when using human blood cells. Increasing the cell concentration more than 5 times the recommended concentration decreases the test signal and therefore decreases the discrimination of the ALDHbr population. However, different cell types can produce different results. Cell titration experiments may be required to determine the optimal cell concentration for different cell types. To stain a large number of cells, it may be better to increase the volume of sample and reagent.
Optimal test performance can be achieved with leukapheresis and peripheral blood samples anticoagulated with acid citrate dextrose (ACD), ethylenediaminetetraacetic acid (EDTA), or sodium heparin. The bone marrow should be anticoagulated with sodium heparin. Cord blood units can be collected in citrate-phosphate-dextrose anticoagulant.
The large numbers of red blood cells present in peripheral blood, apheresis specimens, bone marrow, and umbilical cord blood can compete with stem/progenitor cells for ALDEFLUOR™ substrate. For optimal test performance, lyse red blood cells by treating samples with ammonium chloride. It is necessary to optimize the relationship between lysis buffer and cell count or blood volume (10 to 40 parts of buffer per sample), and time (10 to 30 minutes) and temperature (RT or 2 to 8 °C) of incubation should be optimized. carefully controlled for each lysis buffer and sample type.
Optimal lysis of erythrocytes can be achieved with buffers containing:
• ammonium chloride (e.g., 0.17 M NH4Cl, 10 mM Tris HCl, 0.25 mM EDTA),
• 1 x ABC-Lysepuffer (eBioscience, San Diego, CA)
• VitaLyse® (BioE, St. Paul, MN).
We do not recommend using the following or any other solution that contains a fixer, as this will render the cells unusable:
• CyLyse® (Partec GMBH, Munster, Alemania),
• FACS™-Lyselösung (BD Biosciences, San José, CA.)
NO. ALDEFLUOR™ reagent is a substrate for the enzyme aldehyde dehydrogenase. ALDEFLUOR™ is a marker of viability as the substrate is taken up, catalyzed and retained only by viable cells. Care must be taken to ensure that the reagents used for lysis of erythrocytes do not contain any fixative.
ALDEFLUOR™ has been extensively tested on fresh and cryopreserved cord blood, peripheral blood, and leukapheresis samples from patients and mobilized donors. When done correctly, cryopreservation and thawing should not result in loss of cell viability or fluorescence intensity of ALDHbr cells. Because only viable cells retain the product of the ALDEFLUOR™ reaction, a loss of viability is reflected by a decrease in the percentage of ALDHbr cells and an increase in the percentage of dead/dead cells (detectable by propidium iodide staining). or other stains). ) viability).
The proprietary ALDEFLUOR™ Assay Buffer was developed to optimize the detection of ALDH (or ALDHbr)-positive cells in human blood. The buffer contains an inhibitor of ATP-binding cassette (ABC) transport that prevents active efflux of the ALDEFLUOR™ product from these cells. This transport inhibitor cannot prevent the egress of other types of tissues or other species. Therefore, when using samples other than human blood, reacted cells should be kept at 2-8°C after incubation with activated ALDEFLUOR™ Reagent at 37°C to avoid leakage and consequent loss of fluorescence. . For a list of additional efflux inhibitors that can be added to ALDEFLUOR™ Buffer, see the question "Can I add other efflux inhibitors to ALDEFLUOR™ Assay Buffer?".
The specific product of the ALDH gene, which is expressed in non-human and non-blood products, should not be inhibited by DEAB. The lack of difference between the test and negative control samples may indicate that the inhibitor was not effective or that there is no ALDH activity in the sample cells. Kinetic studies (progressive increase in ALDEFLUOR™ fluorescence in the Negative Control tube with reaction time) may be helpful in distinguishing between these two alternatives. Other ALDH inhibitors can be used as appropriate for the enzyme isoform being expressed. For example, disulfuram inhibits several mammalian ALDH gene products.
When staining non-blood products, it may be necessary to titrate ALDEFLUOR™ Substrate to determine the optimal concentration. We recommend a concentration range of 5 times less to 10 times more than the standard concentration. During the titration, we recommend keeping the DEAB concentration at a 10-fold molar excess of the activated ALDEFLUOR™ reagent and therefore it is necessary to adjust the amount of DEAB when titrating the substrate.
Yes, the lateral population assay can be performed in conjunction with the ALDEFLUOR™ assay (Pearce and Bonnet. Exp Hematol 35: 1437-1446, 2007). The side population test should be performed first, followed by the ALDEFLUOR™ test. We recommend adding 50 µM verapamil to ALDEFLUOR™ Assay Buffer when performing both assays.
ALDEFLUOR™ substrate is a nonpolar fluorescent molecule that diffuses freely in all cells. In the DEAB-treated control, fluorescence reflects the size of the intracellular substrate pool. Fluorescence in the test sample further reflects ALDH activity. Human stem and progenitor cells typically have more ALDH activity than mature cells, and this quantitative difference allows stem cells to separate from other cells.
We recommend washing cells with ALDEFLUOR™ Assay Buffer after reagent reaction to remove background fluorescence from excess substrate. ALDEFLUOR™ reagent shows an emission spectrum similar to FITC with an emission peak at 512 nm Due to the spectral overlap of ALDEFLUOR™ reagent with fluorochromes detected below 650 nm, we recommend the use of fluorochrome-conjugated antibodies, which are found in the emission higher wavelengths for antigens that normally have low levels of expression. For example, when studying the co-expression of CD34 in ALDHbr cells, we used the combination of antibodies, CD45-phycoerythrin (PE), 7-aminoactinomycin D (7-AAD), and CD34-allophycocyanin (APC). Due to the brightness of the ALDEFLUOR™ reagent fluorophore, we strongly recommend using compensating controls for each experiment. Adequate compensation is not achieved with commercially available fluorescent beads.