Cell quest pro version 6

DNA damage was assessed by the phosphorylation of the histone variant H2AX (γH2AX) using FlowCellect^TM 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 previously^{30 (link)}.

hBM-MSC immunophenotypes were evaluated during long-term culture^{25 (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).

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.

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).

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.

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)].