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, 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 (29 mentions)
Facsaria, by BD (20 mentions)
Facsaria 2, by BD (19 mentions)
Facscanto 2, by BD (15 mentions)
Facsaria 3, by BD (14 mentions)
Facscalibur flow cytometer, by BD (13 mentions)
Facsdiva software, by BD (10 mentions)
Gallios flow cytometer, by Beckman Coulter (9 mentions)
Cytoflex flow cytometer, by Beckman Coulter (9 mentions)
Lsrfortessa, by BD (9 mentions)
Accuri c6, by BD (8 mentions)
Accuri c6 flow cytometer, by BD (8 mentions)
Facsaria flow cytometer, by BD (8 mentions)
Fortessa, by BD (8 mentions)
Propidium iodide, by Merck Group (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)
Propidium iodide, by Thermo Fisher Scientific (6 mentions)
608 protocols using aldefluor kit
1
Isolation of Melanoma Stem Cells by ALDH Activity
2
ALDEFLUOR Assay for ALDH+ Cell Detection
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The assay was performed using ALDEFLUOR kits (Stem Cell Technologies Inc., Canada) according to the manufacturer's instructions. Briefly, BODIPY‑aminoacetaldehyde is a non‑toxic fluorescent ALDH substrate able to freely diffuse into intact and viable cells. It is degraded by ALDH into BODIPY‑aminoacetate which is fluorescent and remains inside the cells. The fluorescence intensity is proportional to the ALDH activity when DEAB, the ALDH1 inhibitor is used to control background fluorescence. The fluorescence intensity was examined by a flow cytometer (Accuri C6, BD Biosciences, USA). For ALDH+ cell sorting, a FACSAria III flow cytometer (BD Biosciences, USA) was used.
3
Identification of Tumor-Initiating Cells
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Cells were stained and sorted as previously described,12 (link) using BD LSRII and FACSAriaII hardware, and analyzed by FACSDiva (BD Bioscience, San Jose, CA, USA) or FlowJo (FlowJo LLC, Ashland, OR, USA) software. Zombie Yellow Fixable Viability kit, anti-mouse PD-L1 (Bv421, clone 10F.9G2), CD44 (Per-CP-Cy5), CD133 (PE-Cy7), CD24 (PE), anti-human CD44 (Per-CP-Cy5), CD24 (PE), PD-L1 (PE-Cy7, clone 29E.2AE) and matched isotype controls were purchased from BioLegend (San Diego, CA, USA). ALDEFLUOR kits were purchased from STEMCELL Technologies (Vancouver, BC, Canada). The ALDEFLUOR assay was done with 1×106 ES2 cells per ml. A volume of 5 μl of the specific aldehyde dehydrogenase (ALDH) inhibitor diethylaminobenzaldehyde was added to control tubes. Tubes were incubated with 5 μl BODIPY aminoacetaldehyde, a fluorescent substrate for ALDH and incubated for 45 min at 37 °C. Following washes, samples were kept at 4 °C for remaining staining. TICs were defined as CD44+CD133+CD24+ (B16)13 (link), CD44+CD24+ (ID8)14 (link) and ALDHhi or CD44+CD24− (as indicated, ES2)15 (link),16 (link) by flow cytometry.
4
Measuring Aldehyde Dehydrogenase Activity
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Aldehyde dehydrogenase activity was measured using ALDE-FLUOR kits (STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer’s instructions. For details, see Supplementary Materials and Methods.
5
ALDH-positive Cardiac Progenitor Cells
6
Isolation and Characterization of ALDH+ Cells
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For the separation of aldefluor‐positive (ALDH+) and ‐negative (ALDH−) cells, ALDEFLUOR™ kits (STEMCELL Technologies, Cologne, Germany) were used as recommended by the manufacturer. Five percent of the cells with the highest (ALDH+) or the lowest ALDH1‐activity (ALDH−) respectively were fluorescence activated cell sorting (FACS) sorted (FACS Aria® Cell Sorter; Becton Dickinson, Hamburg, Germany). The effects of forced OLFM4 overexpression on ALDH activity were measured by applying ALDEFLUOR™ kits in combination with an Accuri C6 flow cytometer (Becton Dickinson) and C‐Flow Plus Software. For the exclusion of dead cells, 1 μg/ml propidium iodide (Sigma‐Aldrich) was added. Cutoff values for aldefluor were determined by addition of the ALDH1‐inhibitor diethylamino‐benzaldehyde.
7
Quantifying Stem Cell Markers
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Cells (2 × 105) were counted and incubated in flow buffer (2% FBS in PBS) containing CD133 antibody (BD Biosciences, USA) for 20 min at 4°C. Unbound antibodies were washed with flow buffer and cells were analyzed using a FACS SCAN (BD Biosciences, USA). ALDEFLUOR kits (StemCell Technologies, USA) were used to determine increased ALDH enzymatic activity. Cells (2 × 105) cells were suspended in ALDEFLUOR assay buffer containing ALDH substrate and incubated for 30 min at 37°C. Intracellular fluorescence was measured by flow cytometry using a FACS SCAN and Cell Quest ProTM software. All experiments were performed in triplicate and repeated three times.
8
Identifying Stem-like Tumor Cells by ALDH Activity
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The stem-like tumor cells with highly active ALDH were identified using ALDEFLUOR kits (Stem Cell Technologies, cat. #01700), according to the manufacturer’s instructions. Briefly, 1 × 105 cells were incubated with ALDEFLUOR assay buffer supplemented with ALDH substrate. An aliquot of cells exposed to ALDEFLUOR assay buffer was treated with a specialized ALDH inhibitor (diethylaminobenzaldehyde, DEAB) under the same conditions; this was set as a negative control. After incubation at 37 °C for 40 min, the fluorescence intensity was measured via FACS analysis.
9
Immunoblotting and Cell Invasion Assay
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Protein A/G beads were obtained from Santa Cruz Biotechnology (San Diego, CA). Polyclonal anti-phospho-p38 MAPK (Thr180/Tyr182) antibody was purchased from Life Technologies (Carlsbad, CA) and Cell Signaling Technology Inc. (Beverly, MA). Anti-p38α, p38β, p38γ MAPK and RhoC antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA). Anti-GAPDH antibody was obtained from Research Diagnostics, Inc. (Concord, MA). MTT assay kit was purchased from Roche Molecular Biochemicals (Indianapolis, IN). p38γ shRNA, control shRNA, RhoC siRNA and control siRNA were purchased from Santa Cruz Biotechnology (San Diego, CA). Matrigel Matrix basement membrane and Matrigel Invasion Chambers were purchased from BD Biosciences (Bedford, MA). Transwell was obtained from Costar Corp. (Acton, MA). ALDEFLUOR kits were purchased from Stemcell Technologies (Vancouver, Canada). Antibiotic-Antimycotic (Anti-Anti) and cell culture mediums were obtained from Gibco (Life Technologies). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).
10
Quantification of Aldehyde Dehydrogenase Activity
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ALDEFLUOR kits (Stem Cell Technologies, Vancouver, Canada) were used following the manufacturer’s instructions. HCC cells were suspended in Aldefluor assay buffer containing ALDH substrate (BAAA, BODIPY amino acetaldehyde, 1 mmol/L) at 1×106 cells/mL for 30 m, with or without the specific ALDH inhibitor diethylamino benzaldehyde (1 mmol/L). DEAB was used as an internal negative control for each individual experiment to distinguish between high ALDH activity (ALDH positive) cells and cells with low ALDH activity (ALDH negative). Analysis and sorting were conducted fluorescence-activated cell sorting. Aldefluor was excited at 488 nm and fluorescence emission was detected at 530/30. The data were analyzed by Cell Quest Pro and FlowJo (Ashland, KY, USA).
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