Cells were fixed using 70% alcohol and stained with propidium iodide (Sigma) for cell cycle analysis. ALDH1 enzyme activity in viable cells was determined using a fluorogenic dye‐based Aldefluor assay (Stem Cell Technologies, Grenoble, France) according to the manufacturer's instructions. The prepared cells were analyzed by flow cytometry using BD FACS Calibur (BD Biosciences) and cellquest pro software version 3.3 (BD Biosciences) as described previously [22 (link), 23 (link), 26 (link), 27 (link)].
Cell quest pro software version 3
Manufactured by BD
Sourced in United States
The Cell Quest Pro Software Version 3.3 is a software application designed for flow cytometry data acquisition and analysis. It provides a platform for collecting, processing, and visualizing data from flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur flow cytometer, by BD (4 mentions)
Facscalibur, by BD (2 mentions)
Aldefluor assay, by STEMCELL (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc propidium iodide kit, by BD (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
7 protocols using cell quest pro software version 3
1
Cell Cycle Analysis and ALDH1 Enzyme Activity
2
Characterization of Stem Cell Surface Markers
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Fourth passage cells (1.5 × 106) were washed with FACS buffer (1× phosphate-buffered saline, 5 % FBS, 0.1 % sodium azide), and diluted in 1.5 ml of phosphate-buffered saline. Next, PE-conjugated mouse anti-human CD146, CD73, CD29, and HLA-DR, FITC- conjugated mouse anti-human CD34, CD90, CD45, CD13, and CD31, and APC-conjugated mouse anti-human CD105, CD14, and CD44 antibodies were prepared in dark (all from BD Biosciences, USA, except for the monoclonal antibody against human CD105, which was purchased from R&D Systems, USA) and utilized. In each FACS tube, 100 μl of cells was mixed with 10 μl of the corresponding antibody, and incubated for 30 minutes in the dark at 4 °C. The expressions of cellular markers were assessed using a Becton Dickinson FACSCalibur Flow Cytometer (BD Biosciences, USA), and the resulting data were analyzed using Cell Quest Pro Software Version 3.3, BD bioscience, USA).
3
FACS Analysis of ADSCs and MDCs
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ADSCs or MDCs (passage 2) were prepared for fluorescence-activated cell sorting (FACS) analysis by resuspending them at 1 million cells/100 μL in blocking buffer (10% FBS in PBS). For surface marker analysis, ADSCs were incubated with primary antibodies (1:10, dilution in 100 μL of blocking buffer) for 30 minutes (Table 1 ) and then washed twice with 10% sodium azide in blocking buffer. Cells were then incubated with anti-mouse secondary antibodies made in either goat or horse and conjugated to a fluorescein isothiocyanate fluorophore for 30 minutes, washed twice in blocking buffer, and postfixed using 2% PFA in blocking buffer for 15 minutes. For nuclear marker analysis, ADSCs and MDCs were prepared by fixing with 2% PFA in blocking buffer and then permeabilized with permeabilization buffer consisting of 0.2% bovine serum albumin +0.15% Triton-X-100 in Tris-buffered saline. Cells were then incubated with primary antibodies (Table 1 ) for 1 hour and followed by washing twice with block buffer and incubation with anti-mouse secondary antibodies made in either goat or horse and conjugated to fluorescein isothiocyanate fluorophore for 30 minutes. Prepared cells were acquired by BD FACSCalibur flow cytometer (BD Biosciences) using 488 nm laser and analyzed with CellQuest Pro software version 3.3 (BD Biosciences).
4
Aldehyde Dehydrogenase Activity Assay
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The Aldefluor kit (Stem Cell Technologies, Vancouver, BC, Canada) was used to determine the percentage of cells with high Aldehyde Dehydrogenase (ALDH) enzymatic activity. Briefly, 106 cells were resuspended in Aldefluor assay buffer containing ALDH substrate as recommended by the manufacturer. As a negative control for all samples, an aliquot of “Aldefluor-exposed” cells was immediately quenched using an ALDH inhibitor, diethylaminobenzaldehyde (DEAB). After 30 minutes of incubation at 37°C, the cells were centrifuged and resuspended in 500 μL Aldefluor buffer and analyzed using BD FACS Calibur flow cytometer (BD Biosciences). Aldefluor staining was detected within the green fluorescence channel FL1. Samples treated with the inhibitor DEAB (+DEAB) were used as controls to establish the gates defining the ALDH+ region. Cell Quest Pro Software Version 3.3 (BD Biosciences) was used to analyze the data.
5
Annexin V-FITC/PI Apoptosis Assay
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Cell apoptosis was assessed using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (BD Biosciences). PANC-1/GEM cells were seeded into 6-well plates (5x105 cells/per well). Following incubation with GEM (80 µM) or PBS at 37˚C for 48 h, PANC-1/GEM cells were washed with ice-cold PBS and resuspended in binding buffer, followed by the addition of Annexin V-FITC and PI, according to the manufacturer's protocol. Following incubation at 37˚C for 15 min in the dark, the apoptotic cells, including early apoptotic cells and late apoptotic cells, were analyzed with the FACScan flow cytometer (BD Biosciences) and CellQuest Pro software version 3.3 (BD Biosciences).
6
Cell Cycle Analysis and Aldehyde Dehydrogenase Activity
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Cells were fixed using 70% alcohol and stained with propidium iodide (Sigma, St. Louis, MO, USA) for cell cycle analysis. ALDH1 enzyme activity in viable cells was determined using a fluorogenic dye-based Aldefluor assay (Stem Cell Technologies, Grenoble, France) according to the manufacturer’s instructions. The prepared cells were analyzed by flow cytometry using BD FACS Calibur (BD Biosciences, Franklin Lakes, NJ, USA) and CellQuest Pro software version 3.3 (BD Biosciences) as described previously [21 (link),22 (link)].
7
Characterization of hDPSC Immunophenotype
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FACS buffer (1 × phosphate-buffered saline (PBS), 5% FBS, 0.1% sodium azide) was used to wash hDPSCs (1.5 × 10 6 ), which were then diluted in 1.5 ml of PBS. Then, in the dark, we prepared and used FITC-conjugated mouse anti-human CD90, CD45, and CD34 and APC-conjugated mouse anti-human CD44 antibodies (BD Biosciences, USA). 100 μl of cells were combined with 10 μl of the matching antibody in each FACS tube and incubated for 30 min at 4 • C in the dark. Cellular marker expression was measured using a Becton Dickinson FACS Calibur Flow Cytometer (BD Biosciences, USA), and the data was processed using Cell Quest Pro Software Version 3.3 (BD Biosciences, USA) (Ajlan et al., 2015) .
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