Propidium iodide

TUNEL assay was performed as previously described (Tesei et al., 2007 (link)). Briefly, cells were fixed in 1% formaldehyde in PBS on ice for 15 min, suspended in 70% ice cold ethanol and stored overnight at 20°C. Cells were then washed twice in PBS and re-suspended in PBS containing 0.1% Triton X-100 for 5 min at 48°C. Thereafter, samples were incubated in 50 μL of solution containing TdT and FITC conjugated dUTP deoxynucleotides 1:1 (Roche Diagnostic GmbH, Mannheim, Germany) in a humidified atmosphere for 90 min at 37°C in the dark, washed in PBS, counterstained with propidium iodide (2.5 μg/mL, MP Biomedicals, Verona, Italy) and RNAse (10 kU/mL, Sigma–Aldrich) for 30 min at 48°C in the dark and analyzed by flow cytometry. Flow cytometric analysis was performed using a FACS Canto flow cytometer (Becton Dickinson, San Diego, CA, United States). Data acquisition and analysis were performed using FACSDiva software (Becton Dickinson). Samples were run in triplicate and 10,000 events were collected for each replicate.

Bacterial viability was analyzed using flow cytometer (BD FACS verse). 1 mL of P. aeruginosa cultures were incubated separately with coverslip/wing for 30 min, 1 h, 2 h, 7 h, 24 h and 48 h under static condition in 48-well plate. After incubation time, 1 mL of the cell suspensions were transferred to fresh 2 mL centrifuge tubes. Coverslip and wing were sonicated (Lab companion ultra Sonic cleaner UCP-02) to detach the cells from coverslip/wing in 0.5 mL PBS. Post-sonication, coverslip/wing was discarded and the contents were pooled to their respective 1 mL cell suspensions that were collected earlier. Cells attached to the coverslip/wing were pooled to the suspension to get cell concentration of 10⁷, which is the minimal cell count required for analyses using flow cytometry. To this pooled suspension, 10 µM propidium iodide (MP Biomedicals 195458) was added, mixed and incubated in dark for 10 min. propidium iodide stains the dead cells red. The viable cells remain unstained. The suspensions were loaded onto the BD FACS verse flow cytometer and medium speed was selected to analyze the cells. 10,000 events were studied. The results were obtained using BD FACSuite software application. The flow cytometry data obtained are presented in percentage and not in log scale, as it likely eliminates the low-intensity data due to signal compensation (Herzenberg et al. 2006 (link)).

The cells were seeded at 3 × 10⁵ cells per well into 6-well plates and incubated overnight at 37 °C in the carbon dioxide incubator. The cells were then washed with PBS and treated with a Medium as control or with EGCG (10 μM), EGCG (20 μM), BLM (10 μM), BLM (20 μM) or the combinations (10 μM EGCG plus 10 μM BLM), (20 μM EGCG plus 20 μM BLM) for and incubated for 24, 48, or 72 h. The cells were washed with phosphate-buffered saline (PBS), trypsinized, and incubated in ice-cold ethanol for 2 h. After incubation, the cells were washed with PBS and suspended in PBS plus 0.1 % Triton X-100 plus 100 μg/mL RNase A (Sigma Aldrich) plus 40 μg/mL propidium iodide (MP Biochemicals, Salon, OH) for 30 min in the dark. The cells were run on a Coulter Elite Flow Cytometer. propidium iodide, when bound to nucleic acids, has an excitation maximum at 535 nm and an emission maximum at 617 nm. ModFit LT, version 3.0, software (Verity Software, Portland, ME) was used to analyze and categorize cell populations into cell cycle phases.

After 6d of β- or γ-actin depletion by corresponding shRNAs MDA-MB-231 cells were collected for cell cycle analysis. The cells were harvested, washed with PBS, and fixed with ice-cold 80% ethanol, incubated overnight at 4 °C and stored at 4 °C until analyzed by flow cytometry. Briefly, the cells were spun down to remove the ethanol, washed with PBS and stained with 30 µg/mL Propidium Iodide (MP Biomedicals, Strasbourg, France) and 10 ng/mL RNAse A (Fermentas, Vilnius, Lithuania) in PBS for 45 min at 37 °C in the dark. Cells were analyzed using Beckman Coulter Cytomics FC500 flow cytometer and cell cycle distribution was analyzed with ModFit LT (Verity Software House) software.
The quantitative ratio of cells in different mitotic phases was analyzed by cellular staining for DNA by DAPI. Different mitotic phases were observed by microscopic analysis by scoring 300 cells for each condition.

The cell cycle was evaluated using propidium iodide (PI) (MP Biomedicals, Solon, OH, USA) staining and FCM analysis. Adherent PD-neurospheres were harvested, fixed in 70% ethanol for at least one hour and stained with a solution containing 50 μg/ml PI and 75 KU/ml ribonuclease (RNase) (Sigma) in PBS 1X for 30 minutes at room temperature. Twenty thousand events per sample were acquired. The percentages of the cell cycle distribution were estimated on linear PI histograms by using the MODFIT software.