It was demonstrated that ALDH was involved in maintaining the subpopulation of cancer stem-like cells in B16–F10 melanoma cells21 (link). It has been established that the Aldefluor® kit (Stem Cell Technologies) can be used to isolate stem cells with high ALDH activity in many cancer types. In melanoma, we7 (link) and Luo Y, et al22 (link) reported that the sorted population with high ALDH activity was enriched for melanoma stem cells. The ALDEFLUOR kit (Stem Cell Technologies, Vancouver, Canada) was used to isolate cancer stem cells expressing high levels of ALDH from the B16–F10 melanoma cells. For the assay, briefly, cells were suspended in Aldefluor® assay buffer containing BODIPY-aminoacetaldehyde and incubated at 37°C for 30 minutes. Control samples were incubated with the buffer containing 50mM diethylaminobenzaldehyde (DEAB), an ALDH inhibitor. Cell sorting was conducted using a FACSAria flow cytometer with FACSDiva software (BD Immunocytometry). The Aldefluor® staining was detected using the FITC channel. To prevent cross-contamination between ALDHhigh and ALDHlow cells, sorting gates of these 2 populations were set up at least one log apart. The purity of sorted populations was re-analyzed using ALDHhigh and ALDHlow cells and was shown to be greater than 95% ALDHhigh and ALDHlow respectively.
Aldefluor kit
Manufactured by STEMCELL
Sourced in Canada, United States, France, China, United Kingdom, Germany, Japan, Italy
The ALDEFLUOR kit is a laboratory tool used for the identification and isolation of cells with high aldehyde dehydrogenase (ALDH) activity. It provides a standardized method for the analysis and sorting of ALDH-bright cells from cell samples.
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Lab products found in correlation
Facscalibur, by BD (32 mentions)
Facsaria, by BD (20 mentions)
Facsaria 2, by BD (19 mentions)
Facscanto 2, by BD (16 mentions)
Facsaria 3, by BD (14 mentions)
Facscalibur flow cytometer, by BD (13 mentions)
Facsdiva software, by BD (11 mentions)
Lsrfortessa, by BD (10 mentions)
Gallios flow cytometer, by Beckman Coulter (10 mentions)
Accuri c6 flow cytometer, by BD (9 mentions)
Accuri c6, by BD (9 mentions)
Cytoflex flow cytometer, by Beckman Coulter (9 mentions)
Facsaria flow cytometer, by BD (8 mentions)
Fortessa, by BD (8 mentions)
Propidium iodide, by Merck Group (7 mentions)
Cellquest software, by BD (6 mentions)
Macsquant analyzer, by Miltenyi Biotec (6 mentions)
Facsaria 2 cell sorter, by BD (6 mentions)
Lsrii flow cytometer, by BD (6 mentions)
Facsverse, by BD (6 mentions)
641 protocols using aldefluor kit
1
Isolation of Melanoma Stem Cells by ALDH Activity
2
ALDH Activity Assay Using ALDEFLUOR Kit
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Aldehyde dehydrogenase (ALDH) was assayed using the ALDEFLUORTM kit (Stem Cell Technologies, Vancouver, BC, Canada). Briefly, the ALDEFLUORTM reagent was added and mixed, followed by transferring 0.5 mL of the mixture aliquote to a fresh tube containing 5 μL of ALDEFLUORTM DEAB reagent. After incubation at 37 °C for 30 ~ 60 min, the samples were centrifuged and supernatant was discarded. Cell pellets were resuspended in 0.5 mL of ALDEFLUORTM assay buffer and then analyzed by FACS LSR Fortessa flow cytometer 18 .
3
Quantifying Cellular ALDH Activity
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Cellular ALDH activity was assessed using the ALDEFLUORTM kit (StemCell Technologies Inc.) according to the manufacturer’s procedure. Diethylaminobenzaldehyde, a specific inhibitor of ALDH activity, was used to differentiate cells with low or high ALDH activity. Stained samples (100,000 events/sample) were processed through a cytometer (FACS Calibur, BD Biosciences).
4
ALDEFLUOR Staining for Cell Analysis
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One million cells were stained for 30 minutes at 37°C with ALDEFLUORTM kit (STEMCELL Technologies) according to the vendor's protocol. Cells were subsequently stained with DAPI (0.2 μg/ml, Sigma-Aldrich; 20 min, 4°C). Separate staining for CD133 was performed using anti-mouse CD133 APC antibody (clone 315-2C11, BioLegend) and 0.2 μg/ml DAPI (Sigma-Aldrich) (20 min, 4°C). Cells were analyzed using BD LSRFortessa (BD Bioscience) flow cytometer.
5
Aldehyde Dehydrogenase Activity Assay
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For the determination of aldehyde dehydrogenase (ALDH) activity analysis, we used the ALDEFLUORTM Kit (STEMCELL Technologies, Cambridge, UK) according to the manufacturer’s instructions. In short, 1 × 106 colon cancer cells (DLD1 and HCT116) were first cultured with or without sulfasalazine (SSZ, 50 µM, 48 h) and followed by resuspension in 1 mL ALDEFLUOR buffer. The cells were washed in ALDEFLUOR buffer and maintained at 4 °C throughout the cell staining process. ALDH activity was determined using the fluorescence (FL1) and low side scatter (SCC) channels of a BD FACSCantoTM flow cytometry system (BD Biosciences, CA, USA) and FACSDiva software (v 6.1.2, BD Biosciences, CA, USA).
6
Quantifying ALDH Activity in Cells
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AldefluorTM kit (STEMCELL Technologies) was used to determine ALDH enzyme activity, following company guidelines. Briefly, ALDH reagent (BAAA) freely diffuses into viable cells and ALDH enzyme catalyzes its conversion to BAA, which is negatively charged and retained into cells. Intracellular BAA increases fluorescence which can be analyzed through flow cytometry. As seen before for 12 and 24 h treatments, 0.5xIC50 and 3xIC50 (calculate at 48 h on parental cells) for doxorubicin and paclitaxel, respectively, were used. After treatment, cells were detached and 2 × 105 cells per sample were collected. After washing with PBS, 500 µL of the kit buffer was added to each condition. Afterwards, 2.5 µL of the ALDH reagent was added and 250 µL of the suspension was immediately transferred to a new Eppendorf with 2.5 µL of the ALDH inhibitor N,N-diethylaminobenzaldehyde (DEAB), in order to consider background fluorescence (Supplementary Figure S3 ). All samples were incubated at 37 °C for 40 min. Cells were then centrifuged and washed and samples were prepared to be analyzed through cytometry (FACSCalibur II, BD Bioscience). Images were obtained with FlowJo software (Flow Jo LLC, Ashland, OR, USA.
7
ALDH Activity Evaluation in Cells
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ALDH staining was performed with the ALDEFLUORTM kit (Stem Cell Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions. In brief, cells were suspended at 1 × 106 cells/ml in Aldefluor assay buffer containing ALDH substrate (BAAA, BODIPY aminoacetaldehyde, 1 mmol/l), with or without the specific ALDH inhibitor diethylaminobenzaldehyde (1 mmol/l) for 30 min. Diethylaminobenzaldehyde serves as internal negative control for each individual experiment, and allows to distinguish between ALDH-bright (ALDH positive) cells and cells with low ALDH activity (ALDH negative). Analysis and sorting were conducted on a fluorescence-activated cell sorting: Aldefluor was excited at 488 nm and fluorescence emission was detected at 530/30. Dead cells were excluded by gating on forward and side scatter and eliminating the propidium iodide-positive population. The data were analyzed by Cell Quest Pro and FlowJo (Ashland, OR, USA).
8
Flow Cytometric Analysis of Stem Cell Markers
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Monolayer cells were dissociated with trypsin and blocked in prechilled 2% FBS in PBS for 30 min at 4 °C. Cells were filtered through 70 μm cell strainer and counted. For each sample, 250,000 cells were co-stained with PE-conjugated anti-CD24 and APC-conjugated anti-CD44 or stained with APC-conjugated anti-MUC1 antibodies (BD Bioscience, San Diego, USA) for 30 min on ice. As negative and positive controls, we used non-stained and single-stained cells, respectively. Cells were analysed using Accuri C6 flow cytometer (BD Biosciences) and Flowjo software (Tree Star Inc.).
ALDEFLUORTM Kit (Stemcell Technologies) was used to analyse Aldehyde dehydrogenase (ALDH) enzyme activity, according to the manufacturer’s protocol. A total of 106 cells were incubated with 5 ul ALDH substrate (Bodipy-aminoacetaldehyde) for 45 min at 37 °C. For the negative control, an aliquot of each sample was incubated with diethylaminobenzaldehyde (DEAB). Analysis were performed using Accuri C6 flow cytometer.
ALDEFLUORTM Kit (Stemcell Technologies) was used to analyse Aldehyde dehydrogenase (ALDH) enzyme activity, according to the manufacturer’s protocol. A total of 106 cells were incubated with 5 ul ALDH substrate (Bodipy-aminoacetaldehyde) for 45 min at 37 °C. For the negative control, an aliquot of each sample was incubated with diethylaminobenzaldehyde (DEAB). Analysis were performed using Accuri C6 flow cytometer.
9
ALDH Activity Analysis by Flow Cytometry
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The ALDEFLUORTM Kit (Stem Cell Technologies, Vancouver, BC, Canada) was used to detect intracellular enzyme activity of ALDH. Samples were prepared according to manufacturer’s instructions and ALDH activity was analyzed using BD Canto II Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Dead cells were excluded from the analysis based on 4′,6-diamidino-2-phenyl-indole (DAPI) staining. Data were analyzed by the FCS Express program (De Novo Software, Glendale, CA, USA).
10
ALDH Activity Measurement in TNBC Cells
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TNBC cells were washed once with PBS and harvested using Accutase (BD Biosciences, Franklin Lakes, NJ, USA). ALDH activity was measured using the ALDEFLUORTM kit (STEMCELL Technologies, Vancouver, BC, Canada) and flow cytometry per the manufacturer’s protocol. Treatment with the ALDH inhibitor N,N-diethylaminobenzaldehyde was used as a negative control.
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