ALDEFLUOR™ kit for the identification, evaluation and isolation of stem and progenitor cells expressing high levels of ALDH. ALDEFLUOR&#; Kit from. PRODUCT DESCRIPTION. ALDEFLUOR™ is a reagent kit that is used to identify human cells that express high levels of the enzyme aldehyde dehydrogenase. Here is a quick guide to setting up your FACS analysis with ALDEFLUOR: We’ ve used the Aldefluor kit extensively for both cell lines and for freshly dissociated .
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Cells expressing high levels of ALDH become brightly fluorescent ALDHbr and can be identified and enumerated using a standard flow cytometer or isolated by cell sorting for further purification and characterization. The use of an internal cellular enzyme for identifying and isolating stem and progenitor cells provides an alternative to the more traditional method of staining with antibodies against cell surface antigens.
Safety Data Sheet 1. Safety Data Sheet 2. Safety Data Sheet 3. Safety Data Sheet 4. Safety Data Sheet 5. Educational Materials 21 Brochure. 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. This is not recommended. The assay buffer incorporates an efflux pump inhibitor to produce optimal discrimination of the ALDHbr cells and to maximize fluorescent signal stability.
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. For nonhematopoietic cells optimal incubation times may be different. It is recommended to test different incubation times and determine the optimal incubation time for different cell types.
Yes, but full staining will take at least 3 – 4 hours. These reagents may also improve discrimination of the ALDHbr population, but results will vary by sample type. Sodium azide may be toxic to cells. Ice is the universal efflux inhibitor.
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.
ALDEFLUOR™ Kit | STEMCELL Technologies
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 ACDethylenediaminetetraacetic acid EDTAor 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? For optimal assay performance, lyse the erythrocytes by treating the samples with ammonium chloride. What alfefluor can be used to lyse erythrocytes? Optimal erythrocyte aldeflour can be achieved with buffers containing: We do not recommend use of the following or any aldfluor solution that contains a fixative, as these will render the cells nonviable: Can fixed cells be used with this assay?
It is important to ensure that reagents used for erythrocyte lysis do not contain a fixative. If done correctly, cryopreservation and thawing should not cause loss in cell viability or fluorescence intensity of ALDHbr cells. This transport inhibitor may not prevent efflux from other tissue types or from other species.
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.
Other ALDH inhibitors can be used as appropriate for the enzyme isoform expressed. Alefluor suggest a range of concentrations from 5-fold less to fold more than the standard concentration. Why are all the cells in the cytogram fluorescent to some degree? 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 aldefuor than mature cells, and this quantitative difference allows stem cells to be resolved from the other cells. Adequate compensation will not be achieved with commercially available fluorescent beads. Product Applications This product is designed for use in the following kig area s as part of the highlighted workflow stage s.
Research Area Workflow Stages for. Brain Tumor Stem Cell Research. Hematopoietic Stem and Progenitor Cell Research. Cell Sourcing and Isolation. Mammary Epithelial Cell Research. Tissue Dissociation and Isolation. Cell Culture and Assay. Mesenchymal Stem and Progenitor Cell Research. Neural Stem and Progenitor Cell Research. Prostate Epithelial Cell Research.
Data and Publications Data. Abstract The gastrointestinal tract is continuously exposed to many environmental factors that influence intestinal epithelial cells and the underlying mucosal immune system.
In this article, we demonstrate that dietary fiber and short chain fatty acids SCFAs induced the expression of the vitamin A-converting enzyme RALDH1 in intestinal epithelial cells in vivo and in vitro, respectively.
Furthermore, our data showed that the expression levels of RALDH1 in small intestinal epithelial cells correlated with the activity of vitamin A-converting enzymes in mesenteric lymph node dendritic cells, along with increased numbers of intestinal regulatory T cells and a higher production of luminal IgA.
Moreover, we show that the consumption of dietary fiber can alter the composition of SCFA-producing microbiota and SCFA production in the small intestines. In conclusion, our data illustrate that dietary adjustments affect small intestinal epithelial cells and can be used to modulate the mucosal immune system.
Zhao Q et al. Abstract Mesenchymal stem or stromal cells MSCs have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments.
These issues hinder the potential applications of MSCs, especially those in cancer patients. To circumvent these issues, we derived MSCs from transgene-free human induced pluripotent stem cells iPSCs efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable, but still underwent senescence after prolonged culture and did not form teratomas.
The protocol is scalable and can be used to prepare the large number of cells required for off-the-shelf” therapies and bioengineering applications. Abstract Despite progress in the development of drugs that efficiently target cancer cells, treatments for metastatic tumours are often ineffective. The now well-established dependency of cancer cells on their microenvironment suggests that targeting the non-cancer-cell component of the tumour might form a basis for the development of novel therapeutic approaches.
However, the as-yet poorly characterized contribution of host responses during tumour growth and metastatic progression represents a limitation to exploiting this approach. Here we identify neutrophils as the main component and driver of metastatic establishment within the pre- metastatic lung microenvironment in mouse breast cancer models.
Neutrophils have a fundamental role in inflammatory responses and their contribution to tumorigenesis is still controversial. Using various strategies to block neutrophil recruitment to the pre-metastatic site, we demonstrate that neutrophils specifically support metastatic initiation.
Importantly, we find that neutrophil-derived leukotrienes aid the colonization of distant tissues by selectively expanding the sub-pool of cancer cells that retain high tumorigenic potential. Genetic or pharmacological inhibition of the leukotriene-generating enzyme arachidonate 5-lipoxygenase Alox5 abrogates neutrophil pro-metastatic activity and consequently reduces metastasis.
Our results reveal the efficacy of using targeted therapy against a specific tumour microenvironment component and indicate that neutrophil Alox5 inhibition may limit metastatic progression. Villablanca EJ et al. Nature OCT Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function.
Viale A et al. Abstract Pancreatic ductal adenocarcinoma PDAC is one of the deadliest cancers in western countries, with a median survival of 6 months and an extremely low percentage of long-term surviving patients. Targeting oncogene-driven signalling pathways is a clinically validated approach for several devastating diseases. Still, despite marked tumour shrinkage, the frequency of relapse indicates that a fraction of tumour cells survives shut down of oncogenic signalling.
We demonstrate that a subpopulation of dormant tumour cells surviving oncogene ablation surviving cells and responsible for tumour relapse has features of cancer stem cells and relies on oxidative phosphorylation for survival.
Transcriptomic and metabolic analyses of surviving cells reveal prominent expression of genes governing mitochondrial function, autophagy and lysosome activity, as well as a strong reliance on mitochondrial respiration and a decreased dependence on glycolysis for cellular energetics.
Accordingly, surviving cells show high sensitivity to oxidative phosphorylation inhibitors, which can inhibit tumour recurrence. Our integrated analyses illuminate a therapeutic strategy of combined targeting of the KRAS pathway and mitochondrial respiration to manage pancreatic cancer.