P4864

NCCIT and NTERA-2 cells treated with nocodazole (50 ng/mL) for the indicated times were analyzed by propidium iodide staining (P4864; Sigma-Aldrich) and flow cytometry at the Flow Cytometry Core Facility (National Cancer Center) using FACSVerse (BD Biosciences, San Jose, CA, USA), as described previously [37 (link)].

Cells treated with or without LD100 were constructed as described above. Then, cells were fixed with 70% EtOH at 4°C for 10 h. After washing with PBS (DU145 and HeLa) or D-PBS (RWPE-1), cells were incubated with PBS or D-PBS/0.25% Triton X-100/20 μg/mL RNase A (Invitrogen, 12091021) at 37°C for 1 h. Then, propidium iodide (Sigma-Aldrich, P4864) was added into the above mixture solution and reached the final concentration 30 μg/mL. Finally, cells were incubated with the PI solution at room temperature for 1 h, and the fluorescence intensity was collected by flow cytometry (BD, Accuri™ C6).

Treated EA.hy926 cells on 6-well plates were harvested with trypsin (Catalog #: 25200114, Gibco™, Waltham, MA, USA) for cell cycle analysis using flow cytometry as previously described [46 (link)]. Briefly, after aspirating culture media, trypsinized cells were centrifuged at 750× g for 5 min and then washed once with PBS prior to ethanol fixation (5 mL of ice-cold 75% ethanol in PBS per well for 2 h). The ethanol was removed via centrifugation (300× g for 5 min), and the cell pellets were washed once with cold PBS before being resuspended in 0.5 mL PI solution (50 μg/mL PI (Catalog #: P4864, Sigma-Aldrich, St. Louis, MO, USA), 4 mM Na citrate, 0.1% Triton X-100, 50 μg/mL RNase A (Catalog #: T3018L, New England Biolabs, Ipswich, MA, USA), pH adjusted with NaOH to 7.8). The cells were incubated in the dark for 10 min at 37 °C, and 50 μL of 1.38 M NaCl was added to the 0.5 mL solution in each tube immediately after incubation. The samples were carefully pipetted to achieve a single cell suspension and then analyzed by flow cytometry (CytoFlex LX Digital Flow Cytometry Analyzer 4 Laser System (Beckman Coulter, Brea, CA, USA)) at the University of Manitoba Flow Cytometry Core Facility. On average, >15,000 gated events were analyzed to obtain the percentage of cells in sub G0/G1, G0/G1, S and G2/M phases of cell cycle based on the mean fluorescent intensity of PI.

The cell cycle profile was analyzed based on the cellular DNA content. One million adherent P19 stem cells were harvested, washed with PBS and fixed by adding 70% cold (−20 °C) ethanol. Subsequently, the cells were centrifuged at 850 g to remove the ethanol, washed twice with PBS and finally resuspended in 400 μL of PBS. Propidium iodide (20 μg/mL, P4864, Sigma-Aldrich) and 10 µg/mL RNase cocktail (R5000, Sigma-Aldrich) were added and incubated for 30 min at 37 °C. A total of 20 × 10³ events per sample were analyzed using a Cytoflex S flow cytometer (Beckman Coulter, CA, USA) with excitation and emission wavelengths of 488 nm and 605 nm, respectively. All experimental conducted with a voltage of 673 and the FL3 detector with a bandpass of 602–628. The percentages of cells in G₁/G₀, S, and G₂/M phases were determined using the CytExpert 2.1 software.

To challenge the FA/BRCA pathway, LCLs were submitted to increased concentrations of MMC (0–1000 nM, M0503, Sigma-Aldrich) in fresh medium for a period of 120 h. After this period, cells were resuspended in phophate-buffered saline (PBS)–bovine serum albumin (BSA; 0.05%) containing 0.5 mg/mL propidium iodide (P-4864, Sigma-Aldrich) and incubated for 10 min at 4 °C. Cell viability was determined by flow cytometry based on the PI exclusion test. The analysis was carried out, taking into account the viability of the 0–3 nM cells as a reference in each cell type condition. All the doublets were discarded from the analysis. Flow cytometry analysis was performed on an FACS Calibur (Becton-Dickinson, San Jose, USA). As internal controls, a healthy donor (normal control) was used in each assay, along with three FA patients (positive controls in one of the experiments (kindly provided by Dr Juan Bueren´s laboratory).

Cells were incubated with 10 μM BrdU for 2 hours prior harvest. Cells were then trypsinized and fixed overnight at 4°C in 70% ethanol in calcium- and magnesium-free phosphate buffered saline (PBS). Ethanol solution was removed and cells were incubated in 3 mL of 0.08% pepsin in 0.1 N HCl at 37°C for 20 minutes. Pepsin was removed and nuclei were incubated in 1.5 mL of 2 N HCl at 37°C for 20 minutes. The nuclei containing acid solution was neutralized with 3 mL of 0.1 M sodium borate. Nuclei were spun out of neutralized acid and washed with 2 mL IFA buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 4% FBS and 0.1% sodium azide with 0.5% Tween-20 added on the day of use) and then incubated overnight at 4°C with anti-BrdU clone MoBU-1 conjugated to AlexaFluor488 (Invitrogen B35130) in IFA buffer. DNA was stained for 30 minutes with IFA buffer with of 50 μg/mL propidium iodide (PI) (Sigma-Aldrich #P4864) and 5 μg/mL RNase A (Sigma #R4642). Cell cycle analysis (bivariate plots of BrdU incorporation and DNA content) was performed on a FACSCanto II (Becton Dickinson). Data were collected and analyzed using FlowJo software (FlowJo, Ashland, OR, USA).

The scaffolds were rinsed with PBS three times and then methanol was added in order to fix the adhered cells. They were kept for 20 min at room temperature; thereafter, they were stored at −20 °C. The scaffolds were rinsed three times with PBS. The samples were then incubated at room temperature and in dark with DiOC₆(3) (D273, Life Technologies, Eugene, Oregon) diluted at 1:700 in PBS. After 45 min, the liquid was aspirated and propidium iodide (P4864, Sigma Aldrich, Darmstadt, Germany) in dilution 1:200 in PBS was added for 10 min. Finally, the samples were washed with PBS three times. For scanning, we used a confocal microscope Zeiss LSM 510 DUO (Zeiss, Jena, Germany). Cell nuclei stained with propidium iodide are visualized in red, cytoplasmic membranes in green color. The experiment was carried out in three biological repeats per group. Representative images are presented.