Access resistance as well as fast and slow capacitance were compensated and monitored throughout the recordings. All current measurements were filtered at 2.9 kHz and digitized at PI3K phosphorylation 2 kHz. The cells were held at 0 mV and step
pulses of 400 ms duration were applied from 0 mV to +40 mV every 20 s to monitor the activation of the swelling activated chloride current (IClswell). To establish the current to voltage (IV) relationship of the current, step pulses of 500 ms duration were applied every 10 min from −120 to +100 mV in 20 mV increments from a holding potential of 0 mV. For data analysis, Fit Master (HEKA Elektronik, Germany) and EXCEL (Microsoft, USA) software were used. Current values were normalized to the membrane capacity to obtain the current density. For assessment of apoptosis and cell size analysis, cells were seeded in 100 mm diameter
Petri dishes at a density of 100,000/ml (HEK29 Phoenix cells) or 120,000/ml (HT-29 cells) and grown for XL184 research buy 19 h (HEK29 Phoenix cells) or 22 h (HT-29 cells) in the presence of 0.1, 0.5, 1.0, 5.0, or 10 μM curcumin (HEK29 Phoenix cells), 5.0, 10, 20 or 50 μM curcumin (HT-29 cells) or 0.05% DMSO as the vehicle. Cells treated with 20 μM staurosporine (Sigma, Austria) for 4 h served as positive controls for apoptosis. Cells were harvested using accutase (Sigma, USA), centrifuged and washed twice in binding buffer (in mM: NaCl 140, CaCl2 2.5, HEPES 10, pH 7.4). 1 × 106–2 × 106 cells/sample were stained with FITC-conjugated Annexin-V (ImmunoTools, Germany) for 20 min. After two washes with binding buffer, 5 μl of 7-AAD (7-AMINI-ACTINOMYCIN D) viability stain solution (BioLegend, Inc.) was added to each sample (final volume 0.5 ml). After 15 min, cells were used for flow cytometry. All preparation medroxyprogesterone steps were performed at room temperature in the dark. Fluorescence
emissions of FITC-Annexin-V on FL-1 (525 nm band pass filter) and 7-AAD on FL-3 (670 nm long pass filter) were measured upon excitation with a 488-nm argon laser using a Cell Lab Quanta™ SC flow cytometer (Beckman-Coulter). Unstained and single-stained samples were used for setting the electronic volume (EV) gain, FL-1 and FL-3 PMT-voltages and for compensation of FITC-spill over into the 7-AAD channel. Debris (particles diameter < 7 μm) and cell aggregates (>20 μm) were excluded from analysis. 20,000–30,000 single cells (diameter 7–20 μm) were analyzed in each sample and depicted in FL-3 versus FL-1 dot plots. Quadrant regions were set to segregate cells in four different populations: 7-AAD–/Annexin-V – cells were considered as non-apoptotic, 7-AAD−/Annexin-V+ cells as early apoptotic, 7-AAD+/Annexin-V+ cells as late apoptotic, and 7-AAD+/Annexin-V – cells as post-late apoptotic/necrotic.