OV90 and PEO1 cells were used as ALDH1A1 and ALDH 1A3 high expression cell lines, respectively. Cells were grown and ALDEFLUOR (STEMCELL Technologies) assays performed as previously described.22 (link) Briefly, cells were cultured to 70-80%, trypsinized, washed with PBS, and then re-suspended in ALDEFLUOR buffer. ALDEFLUOR reagent was added, cells were rapidly mixed and then equally distributed into tubes containing inhibitor, DEAB control, or vehicle. After a 30 minute incubation at 37 °C, cells were washed with ALDEFLUOR buffer and maintained on ice until flow-cytometric analysis. Gating was based on DEAB (inhibitor control- set to <1%) and vehicle control treated cells (positive control). The percent of ALDH inhibition was set as percentage of ALDEFLUOR positive cells for a particular sample and the percentage of ALDEFLUOR positive cells vs. vehicle treated control. The Two-way ANOVA with Tukeys multiple comparison test (Prism GraphPad) was used to determine statistical significance between samples treated with compound or vehicle.
Aldefluor
Manufactured by STEMCELL
Sourced in Canada, United States, France
The ALDEFLUOR Kit is a fluorescent-based assay used to identify and isolate cells with aldehyde dehydrogenase (ALDH) enzymatic activity. ALDH is a marker associated with stem and progenitor cells. The kit provides reagents and protocols to measure ALDH activity in live cells.
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57 protocols using aldefluor
1
ALDH1A1 and ALDH1A3 Expression Assay
2
Quantifying Tumor-Initiating Cells via Aldefluor Assay
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To determine the population of TICs, we performed Aldefluor assays (Stem Cell Technologies, Vancouver, British Colombia) according to manufacturer’s recommendations and as previously described [12 (link)]. Briefly, 1 × 106 cells were plated in a 10 cm dish overnight prior to 24-hour treatment with A69 (10 μM). After A69 treatment, 2 × 105 cells were suspended in Aldefluor assay buffer containing ALDH substrate (Bodipy-Aminoacetaldehyde or BAAA), which served as the “test” sample. As a control, half of this sample was moved to a second tube where diethylaminobenzaldehyde (DEAB), a specific ALDH1 enzyme inhibitor was also added. Both samples were incubated for 60 minutes at 37°C. The fluorescent ALDH-expressing cells were detected in the green channel (515–535 nm) of a Cytotomics FC 500 (Beckman Coulter, Brea, CA) flow cytometer. Data were analyzed using FlowJo Flow Cytometry Data Analysis Software (Tree Star, Ashland, OR). The percent shift between gated events in the test versus control samples was calculated to give the relative TIC population.
3
Assessing Progenitor Cell ALDH Activity
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ALDH activity, a conserved progenitor cell function, was assessed by flow cytometry. Using the Aldefluor™ assay (Stem Cell Technologies, Vancouver, BC), as per the manufacturer's instructions. Briefly, 5 μL of activated Aldefluor reagent was added to 1 mL of cell suspension and incubated for 45 minutes at 37°C. Cells were washed and resuspended in 500 μL of ice-cold Aldefluor assay buffer and ALDH activity was measured using flow cytometry. As a negative control, Aldefluor DEAB reagent was used [26 (link)]. Samples were run in triplicate.
4
ALDH Expression in Intestinal DCs
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5
Quantifying Aldehyde Dehydrogenase Activity
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ALDH activity, a conserved progenitor cell function, was assessed by flow cytometry at days 1, 3, 7, and 14 using the Aldefluor™ assay (STEMCELL Technologies), as per the manufacturer's instructions. Briefly, 5 μL of activated Aldefluor reagent was added to 1 mL of cell suspension and incubated for 45 minutes at 37°C. Cells were washed and resuspended in 500 μL of ice-cold Aldefluor assay buffer, and ALDH activity was measured using flow cytometry. As a negative control, Aldefluor™ DEAB reagent was used [26 (link), 31 (link)].
6
Flow Cytometry of Stem Cell Markers
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Following being thawed and washed, cell suspensions were incubated with the ALDEF substrate (Aldefluor®, 1 μM, Stemcell Technologies) for 20 min (human and NHP cells) or 45 min (dog cells), at 37°C. Controls were obtained by prior incubation of cells with 50 mM of the specific ALDEF inhibitor DEAB.48 Cells were centrifuged and kept on ice; surface antigens were detected by incubation with allophycocyanin‐labelled CD34 (BD) or phycoerythrin (PE)‐labelled CD9, CD10, CD29, CD31, CD36, CD44, CD47, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD56, CD61, CD71, CD105, CD106, CD140a, CD140b, CD143, CD146, CD184, and CD309 (3/100, 15 min, 4°C, BD). Cells were analysed using FACSCalibur (BD) and the CellQuest Software (2.103 to 104 events analysed, owing to the small size of the biopsies). Non‐specific fluorescence was determined using negative isotype controls (BD).47 Data were analysed and plotted using GraphPad Prism.
7
Quantifying Tumor-Initiating Cell Frequencies in Lung Cancer
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CAL27- and CAL33-derived CYP1B1-WT and CYP1B1-VAR cells were stained with ALDH reagents (Aldefluor, Stemcell Technologies Inc). ALDH+ and ALDH− cells were sorted with a FACSAria Cell Sorting System (BD Biosciences) and resuspended in 100 μL of 3:1 PBS:Matrigel (BD Biosciences). In vivo studies were then performed; 3,000–300 for CAL27 CYP1B1-WT and 3000–10 for CAL27 CYP1B1-VAR FACS-sorted cells were injected subcutaneously into the flanks of 6- to 8-week-old female NSG mice; the right flank of the mouse received the ALDH+ cells, whereas the left flank received the ALDH− cells. Engrafted mice were inspected once a week by visual observation and palpation for the appearance of tumours. The tumour volume (V) was determined weekly from the length (a) and the width (b) of the tumour, using the formula V = ab2/2. A portion of each tumour tissue was fixed in 10% formaldehyde and embedded in paraffin for IHC analysis. The frequency of tumorigenic cells (estimated with upper–lower limits) was calculated by extreme limiting dilution analysis.16 (link) Mice tumour-free survivals were estimated by the Kaplan–Meier method and compared using the log-rank test.
8
Quantifying ALDH-Positive Tumor Cells
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The p53 SMWC-treated and untreated human and mouse tumor cell lines were analyzed for ALDHpositive/ALDHbrightcellsby flow cytometry using ALDEFLUOR (StemCell Technologies Vancouver, BC, V5Z 1B3, Canada), as previously described [18 (link), 19 (link), 44 (link)]. In general, duplicate aliquots of 2 × 105 tumor cell samples were incubated with ALDEFLUOR, with or without the ALDH inhibitor, diethylaminobenzaldehyde (DEAB) (control), according to the manufacturer's instructions. The control aliquot was analyzed by flow cytometry and set for detection of ≤0.5% ALDHpositive cells. Using this cutoff, the test aliquot was analyzed to identify its ALDHpositive/ALDHbright cell content with ALDHbright cells defined as the ALDHpositive cells with double the mean fluorescence intensity (MFI) of the bulk population of ALDHpositive cells in a sample [19 (link)]. The flow cytometry analyses were performed using a C6-Sample cytometer (BD); all samples were run using identical settings to collect a minimum of 8,000-gated events. Analyses were done using BD CSAMPLER™ ANALYSIS software (BD). The effects of the p53 SMWC on cell growth and percentage content of ALDHpositive/ALDHbright cells were calculated relative to the untreated control cell populations.
9
Characterization of Mammary Cell Subpopulations
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Mammary cells were stained for CD44 and CD24 expression and ALDH activity as described previously (Colacino et al., 2016 (link)). In brief, single mammary cells were first incubated with a lineage-depletion cocktail that consisted of biotinylated antibodies targeted against CD45, HLA-DR, CD14, CD31, CD41, CD19, CD235a, CD56, CD3, CD16, and CD140b (all from eBioscience, except for CD140b [Biolegend] and CD41 [Acris]). Next, cells were stained with Alexa Fluor 750-tagged streptavidin, LIVE/DEAD Fixable Dead Cell Stain (Invitrogen), CD24 (Biolegend), CD44 (Becton Dickinson), and Aldefluor (STEMCELL Technology). Single-color and isotype controls were included for compensation and gating purposes. Aldefluor-positive gating was based on DEAB (negative) controls. Flow-cytometry data analysis was performed with FlowJo software version 10.0.8.
10
Multiparameter Flow Cytometry of Cells
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Single- and multi-color immunostaining was performed according to standard surface and intracellular FACS staining Biolegend protocols (Biolegend, San Diego, CA, USA). Anti-Ep-cam Alexa-Fluor 488 (Biolegend 9c4) was used for identification of epithelial cells. The purity of MF was analysed by staining for CD90-PE (Biolegend Thy1) and α-smooth actin-Alexa Fluor 488 (R&D Systems 1C1420G, Minneapolis, MN, USA) for flow cytometry compared with isotype controls. Anti-G-CSFR-PE (clone lmm741 Biolegend) and G-CSF-FITC (clone 85FSCSF eBioscience, San Diego, CA, USA) were used for the analysis of G-CSFR surface expression and intracellular G-CSF after 4 h exposure to Brefeldin A compared with the isotype controls. Anti-proliferating cell nuclear antigen (PCNA) (clone PCNA01, Biolegend) was used as a proliferation marker. Cancer stem-like populations in carcinoma cell lines were examined by staining with Aldefluor (Stem Cell Technologies, Vancouver, BC, Canada) and anti-CD44-APC (Biolegend clone BJ18) using the manufacturer's Aldefluor staining protocol compared with isotype control. All samples were analysed on a Guava easyCyte 8HT flow cytometer (EMD Millipore, Bellerica, MA, USA), and analysed using FCS Express software (DeNovo Software, Los Angeles, CA, USA).
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