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).
Cell quest pro
Manufactured by FlowJo
Sourced in United States
The Cell Quest Pro is a flow cytometry software designed for data acquisition, analysis, and presentation. It provides users with essential tools for managing and interpreting flow cytometry data. The software supports a range of flow cytometry instruments and offers features for data visualization, gating, and statistical analysis.
Automatically generated - may contain errors
Lab products found in correlation
Aldefluortm kit, by STEMCELL (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Quantikine enzyme linked immunosorbent assay kit, by R&D Systems (1 mentions)
Apc cd314, by BioLegend (1 mentions)
Facscalibur, by BD (1 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
5 protocols using cell quest pro
1
ALDH Activity Evaluation in Cells
2
Cell Cycle and Apoptosis Analysis by Flow Cytometry
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For detection of cell cycle distribution and apoptosis/necrosis, flow cytometry was used as recently described in (Streubel et al,2013 ; Bouchard et al,2018 ). In detail, for quantification of cell cycle distribution, cells were harvested, washed in ice‐cold PBS, and fixed in ice‐cold 70% ethanol overnight. After complete permeabilization, cells were washed twice with FACS buffer. DNA was then stained with 54 µM propidium iodide (PI, Sigma‐Aldrich) in the presence of 38 mM sodium citrate and 250 µg/ml RNase A for 30 min at 37°C in the dark. For quantification of phosphatidylserine on the surface of UVC‐treated cells, 0.5–1 × 106 unfixed cells were incubated with FITC‐labeled Annexin‐V and/or PI (BD Pharmingen) according to the manufacturer´s instructions. All samples were analyzed using BD FACSCalibur flow cytometer (BD Bioscience). Data were processed using the CellQuestPro or FlowJo (FlowJo LLC) Software.
3
Cell Preparation for FACS Analysis
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To prepare cells for FACS analysis, 1×106 (control and treated) cells were washed with 1× PBS, resuspended in 0.5 ml of 1× PBS and incubated with 10 μl of RNase A (10 mg/ml) at 37°C in a water bath for 30 min, followed by addition of 4 μg/ml of Propidium Iodide in dark on ice. Stained cells were analysed on Becton Dickinson FACScan machine and the data were analysed with either CELLQuest Pro or FlowJo software.
4
Quantification of circulating NK cells
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The following antibodies were used to stain peripheral blood mononuclear cell (PBMC) suspensions: fluorescein isothiocyanate (FITC)-CD3, FITC-CD4, peridinin-chlorophyll-protein (PerCP/cyanine 5.5-CD16, phycoerythrin (PE)-CD19, PE-CD56, adenomatous polyposis coli (APC)-CD158 (KIR2D L1/S1/S3/S5), APC-CD158e1 (KIR3DL1), APC-CD159a (NKG2A), APC-CD226 (DNAM-1), APC-CD244 (2B4), APC-CD314 (NKG2D), and Alexa Fluor (AF)647-CD337 (NKp30) (BioLegend, San Diego, CA, USA), eFluor660-CD107a (eBioscience, San Diego, CA, USA), and CD8 (BD Biosciences, Franklin Lakes, NJ, USA). The subpopulation of circulating NK cells was quantified and analyzed using flow cytometry, as described previously.32 (link)
Briefly, 5 × 105 PBMCs after red blood cell lysis were collected for incubation with antibodies for 30 minutes at 4°C, washed with phosphate-buffered saline and fixed with 4% paraformaldehyde for 20 minutes. The cells were then washed twice with phosphate-buffered saline and analyzed using FACSCalibur (BD Biosciences). Data were analyzed using Cell Quest Pro (FlowJo, LLC, Ashland, OR, USA). Human plasma IL-6 and TNF-α levels were measured with commercially available Quantikine Enzyme-Linked Immunosorbent Assay Kits (R&D Systems, Inc., Minneapolis, MN, USA) in accordance with the manufacturer’s instructions.
Briefly, 5 × 105 PBMCs after red blood cell lysis were collected for incubation with antibodies for 30 minutes at 4°C, washed with phosphate-buffered saline and fixed with 4% paraformaldehyde for 20 minutes. The cells were then washed twice with phosphate-buffered saline and analyzed using FACSCalibur (BD Biosciences). Data were analyzed using Cell Quest Pro (FlowJo, LLC, Ashland, OR, USA). Human plasma IL-6 and TNF-α levels were measured with commercially available Quantikine Enzyme-Linked Immunosorbent Assay Kits (R&D Systems, Inc., Minneapolis, MN, USA) in accordance with the manufacturer’s instructions.
5
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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