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18 protocols using cell quest pro version 6

1

Annexin V Apoptosis Assay by Flow Cytometry

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Cells were treated with samples for 72 h and stained with an Annexin V-FITC apoptosis detection kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Briefly, cells were collected after 72 h treatment, washed with PBS, resuspended in 1× binding buffer, and stained with Annexin V-FITC and PI in the dark for 15 min. After incubation, the cells were diluted with 1× binding buffer and analyzed using an FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and BD CellQuestTM Pro version 6.0 software (BD Biosciences, San Jose, CA, USA).
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2

Cell Cycle Analysis by Flow Cytometry

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Cells were seeded and starved in serum-free medium overnight. After starvation, the cells were treated with compounds for the indicated times. After incubation, cells were harvested, washed with PBS, and fixed with 70% ethanol in PBS overnight at −20 °C. The fixed cells were washed with PBS, resuspended in 100 µg/mL of RNase A for 30 min at room temperature, and stained with 50 µg/mL PI. Fluorescence intensity was analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and BD CellQuestTM Pro version 6.0 software (BD Biosciences, San Jose, CA, USA).
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3

PD-L1 Expression after Drug Treatments

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Cells were seeded at 0.5×106 cells per well in 6-well plates for overnight at 37oC in a humidified incubator with 5% CO2. After 24 hours, cells were treated with IC50 value of different drug treatments as described in Figure 2 for 48 hours. After that, expression level of PD-L1 was determined using flow cytometry as follows. Briefly, harvested cells were washed twice with 3% BSA/PBS and incubated with either isotype control or rat anti-PD-L1 (mouse, BioXcell, West Lebanon, NH, USA) for 30 minutes at 4oC. After washing three times, the cells were incubated with anti-rat Alexa Fluor 488 conjugated antibody. The cells washed once more with 3% BSA/PBS and analyzed on FACSCalibur flow cytometer and CellQuest™ Pro version 6.0 software (both from Becton-Dickinson and Co.).
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4

DNA Content Analysis via Flow Cytometry

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For DNA content analysis for all conditions, 106 cells were fixed and permeabilized in 70% ethanol in PBS at −20°C overnight, washed in PBS, treated with RNase (40 U/µl, Boehringer Mannheim) for 20 min at room temperature, and stained with PI (50 µg/ml). Flow cytometric analyses (fluorescence-activated cell sorter (FACS) were performed as described previously (22 (link)) (Becton Dickinson FACScalibur). The used software was CellQuest™ pro version 6.0 (Becton Dickinson).
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5

Quantifying DNA Damage via Flow Cytometry

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DNA damage was assessed by the phosphorylation of the histone variant H2AX (γH2AX) using FlowCellectTM Cell Cycle Checkpoint Ataxia telangiectasia mutated (ATM) DNA Damage Kit (Millipore, Temecula, CA, USA)29 (link). Briefly, a test sample of 1 × 106 cells was fixed, permeabilized and labeled with Alex Fluor 488-conjugated anti-phospho-ATM on ice for 30 min in the dark. DNA was stained with PI at room temperature in the dark for 30 min. At least 20,000 events were analyzed using a FACS Calibur cytometer (BD Biosciences) and ATM enzyme activity and cell distribution in each phase of the cell cycle including sub G1-peak of apoptotic cells were analyzed using CellQuest Pro version 6.0 software (BD Biosciences) as described previously30 (link).
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6

Long-term Immunophenotyping of hBM-MSCs

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hBM-MSC immunophenotypes were evaluated during long-term culture25 (link). Harvested cells were labeled using mouse anti-human monoclonal antibodies: phycoerythrin (PE)-conjugated CD105, CD90, CD73, CD34, CD45, CD11b and CD79a, and fluorescein isothiocyanate (FITC)-conjugated HLA-DR (BD Biosciences, San Jose, CA, USA). As a control, isotype PE-conjugated IgG1 and FITC-conjugated IgG2a (BD Biosciences) were used. The cell suspension containing 1 × 106 cells was incubated with monoclonal antibodies for 15 min at room temperature in the dark and fixed with BD Cytofix (BD Biosciences). The samples were analyzed on FACSCalibur cytometer (BD Biosciences) and the resulting data were processed using CellQuest Proversion 6.0 software (BD Biosciences).
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7

Immunophenotyping of EGFR and ADAM17

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Cells were detached and resuspended in flow cytometry buffer (2/3 (v/v) PBS and 1/3 (v/v) RPMI without additives). For immunostaining, 10 μg/mL α-EREG (goat, polyclonal, R&D Systems, Minneapolis, Minnesota, US) or α-TACE/ADAM17 (mouse, MM0561-8C13, Novus Biologicals, Wiesbaden, Germany) antibody was added to flow cytometry buffer and incubated for 1 h at 4°C. ChromPure goat or mouse IgG (Jackson ImmunoResearch, Cambridgeshire, UK) were used as controls for corresponding primary antibody. After three washing steps, cells were incubated for 1 h at 4°C with FITC-conjugated α-goat (donkey, polyclonal, Jackson ImmunoResearch, Cambridgeshire, UK) or α-mouse (goat, polyclonal, Jackson ImmunoResearch, Cambridgeshire, UK) antibody diluted 1:50 in flow cytometry buffer. Finally, cells were washed three times and resuspended in PI diluted 1:200 in DMEM without additives. The acquisition was performed by FACS Calibur System (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed by Cell Quest Pro™ Version 6 (Becton Dickinson, Franklin Lakes, NJ, USA). To determine the size and granularity of cells the forward (FSC) and sideward (SSC) scatter were used.
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8

Apoptosis Induction Assay in Transfected Cells

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Cells were seeded in 24-well plates and transfected with 0.5 μg expression plasmid. After 24 h, cells were treated with 200 ng/mL Fas ligand (FasL; Immunotools, Friesoythe, Germany), enzalutamide (10 µM final concentration; Hölzel Diagnostika, Cologne, Germany) in androgen-reduced medium or docetaxel (20 nM final concentration; Sigma-Aldrich, Munich, Germany) to induce apoptosis. Cells were collected three days after transfection and stained with Annexin V-FITC (Immunotools, Friesoythe, Germany) diluted 1:40 in 200 µl DMEM for 20 min on ice. Cells were washed with cold DMEM and stained with 200 μL propidium iodide solution diluted 1:200 in DMEM. The acquisition was directly performed utilizing a FACSCalibur System (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed by Cell Quest Pro™ Version 6 (Becton Dickinson, Franklin Lakes, NJ, USA).
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9

CEACAM Expression Profiling in RWPE-1 Cells

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RWPE-1 cells (5×105) were stained with 10 μg/ml anti-CEACAM1 (B3-17, A1B domain), anti-CEACAM1 (1/3/5-Sab, N domain), anti-CEACAM5 (5C8C4), anti-CEACAM6 (1H7-4B), anti-CEACAM20 (1-11A), and anti-CEACAM1/3/5/6/8 (6G5j) monoclonal antibodies (mAb) diluted in 3% FCS/PBS for 1 h at 4°C. In the next step the cells were washed with icecold PBS and incubated with FITC conjugated anti-mouse F(ab’)2 (Dianova, Germany) for 30 min at 4°C. Background fluorescence was determined using isotype-matched Ig mAb. The stained cell samples were examined in a FACScalibur flow cytometer (Becton Dickinson, USA) and analyzed by CellQuest Pro® Version 6 (Becton Dickinson, USA). Dead cells identified by propidium iodide staining (1:200 v/v in 3% FCS/PBS) were excluded from the determination.
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10

FACS Analysis of Apoptosis and Cell Cycle

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The fluorescence-activated cell sorting (FACS) technique was employed to analyze the 3c- and 3e-induced cell death, the activation of executive caspase-3, and the cell cycle in the A549 cells. The quantitative analysis of apoptosis and necrosis was performed using an Annexin V-fluorescein isothiocyanate (FITC)/ propidium iodide (PI) apoptosis kit (BD Biosciences, BD Pharmingen™, San Jose, CA, USA) as previously described [36 (link)]. To determine the active form of caspase-3, the A549 cells were plated into 6-well plates at a density of 6 × 105 cells/well. The next day, the growth medium was replaced with a fresh one supplemented with compound 3c or 3e (10, 20, and 40 µM) or the DMSO vehicle (0.1%; control cells). After 48-h incubation, the samples were harvested and the level of active caspase-3 was determined using the phycoerythrin (PE) Active Caspase-3 Apoptosis Kit (BD Pharmingen™) according to the manufacturer’s instructions. The stained cells were analyzed using FACS Calibur, and data were analyzed using Cell Quest Pro Version 6.0 (BD Biosciences, San Jose, CA, USA) for the Macintosh operating system. The results were calculated as a percent of cells with active caspase-3 among all the analyzed cells. The cell cycle analysis was performed by determination of the DNA contents on the basis of PI staining as previously described [36 (link)].
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