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Aldefluor

Manufactured by BD
Sourced in United States

ALDEFLUOR is a flow cytometry-based reagent system designed to identify and isolate stem and progenitor cells based on the activity of the enzyme aldehyde dehydrogenase (ALDH). The ALDEFLUOR reagent is a fluorescent substrate for ALDH, allowing for the detection and analysis of ALDH-bright cells, which are enriched for stem and progenitor cell populations.

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3 protocols using aldefluor

1

ALDH Activity and CD133 Expression

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ALDH enzymatic activity was assessed as previously described [34 (link)–36 ], using ALDEFLUOR (Stem Cell Technologies, Seattle, WA) according to the manufacturer’s instructions. Following ALDH staining, cells were incubated with CD133-APC antibody (BD Biosciences, Ashland, OR) at 1:20 dilution in ALDEFLUOR buffer for 25 min on ice, protected from light. Cells were washed in PBS and resuspended in 400 µl PBS for analysis on a BD FACSVerse cell analyzer (BD Biosciences, Franklin Lakes, NJ). Cell death was assessed by Annexin V (640,905, Biolegend, San Diego, CA) and propidium iodide (PI) (R37169, Thermo Scientific, Waltham, MA) staining on cells treated as indicated, as previously described [35 , 36 ].
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2

Isolation and Analysis of Tumor-Initiating Cells

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Cells were pretreated and dissociated. For evaluation of the tumor-initiating feature, they were stained with ALDEFLUOR (STEMCELL Technologies, Vancouver, Canada) in presence or absence of the inhibitor diethylaminobenzaldehyde (DEAB; STEMCELL Technologies, Vancouver, Canada) to detect the specific marker ALDH [65 (link)]. In brief, cells were mixed in buffer-diluted ALDEFLUOR, and for control, was immediately transferred to DEAB. After incubation in 37 °C for 30 min, the cells were washed and resolved in the buffer. For ROS, they were labeled with dihydroethidium (Cayman, Ann Arbor, MI, USA). After incubation in 37 °C for 30 min, they were washed and resolved in PBS. The staining results of these cells were then studied and quantified for up to 10,000 cells using fluorescence-activated cell sorting (FACS) with a FACSCalibur system (BD Biosciences, Franklin Lakes, NJ, USA). The data were then analyzed via CellQuest (BD Biosciences, Franklin Lakes, NJ, USA).
The sorting process was through FACSAria™ III (BD Biosciences, Franklin Lakes, NJ, USA) for concomitant isolation of the ALDH-positive and the -negative cells, according to the ALDEFLUOR staining result. The cells were collected in the serum-free, growth factor-supplemented DMEM/F12 medium. At least 20,000 cells were used in each group for immediate RNA preparation, followed by the qPCR as described previously.
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3

Comprehensive Flow Cytometry Analysis

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All flow cytometry analysis such as the NOTCH activity, apoptosis, cell cycle and aldefluor assay were performed using BD FACSCalibur (BD Bioscience). NOTCH activity was examined using a pGreenFire1-Notch plasmid (#TR020PA-1, System Bioscience, Palo Alto, CA, U.S.A.). This lentiviral transfection was performed according to the manufacturer’s protocol. The apoptotic cells were detected using Annexin V-FITC apoptosis detection kit (#C986X37, Sigma Aldrich). Cell cycle phase analysis was carried out using propidium iodide cell staining (#11348639001, Sigma Aldrich) and FlowJo software (ver.10, FLOWJO, Ashland, OR, U.S.A.). To assess ALDH1 activity, we used aldefluor kit (#01700, STEMCELL Technologies, Vancouver, BC, Canada) at a concentration of 50 μlml−1. Diethylaminobenzaldehyde (DEAB) was used to inhibit ALDH1 activity.
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