Gallios analyzer

Flow cytometry analysis was performed on a Gallios analyzer (Beckman Coulter, IN, USA) equipped with violet (405 nm), blue (488 nm), and red (633 nm) solid-state lasers as an excitation source. Purified rat anti-mouse CD16/CD32 was added to the fresh blood samples to block non-antigen-specific binding (Beckton Dickinson, CA, USA). After 5 min incubation at 25°C, fluorochrome-conjugated antibodies (Table S2, Beckton Dickinson, CA, USA) were added to the samples. BD FACS™ lysing solution was then added to lyse red blood cells. Samples were homogenized with vortex and incubated for 5 min at 25°C in the dark. After centrifugation (5 min, 3,220g, 25°C), the supernatant was discarded, and the pellet was resuspended in 400 µL of PBS for immunophenotyping of different White Blood Cells (WBC) for every experimental group (n=6) on days -3, 16, and 18. Events collected from fresh blood mouse samples were displayed in a CD45 vs. side scatter intensity (SS INT) plot to discard debris and define a total WBC population. Every single FACS determination recorded about 150,000 total events, of which 50,000 were CD45 positive. Fluorescence was collected through the corresponding bandpass filters for each indicated surface cell marker. Data were analyzed using KALUZA software (Beckman Coulter, CA, USA).

The percentages of apoptotic cells induced by ARN3236/ARN3261, olaparib, or a combination of both were measured on different ovarian cancer cell lines by FACS using FITC Annexin V/Dead cell Apoptosis kit I (Thermo Fisher Scientific, V13242) according to the manufacturer’s instructions. Briefly, following indicated treatment, cells were harvested and washed once in 1× PBS. Afterward, cells were resuspended in 1× binding buffer containing 5 μL of fluorochrome-conjugated annexin V plus 100 μg/mL propidium iodide. After 15 minutes of incubation at room temperature, cells were centrifuged at room temperature for 3 minutes at 1000g and resuspended in 200 μL 1× binding buffer and analyzed with flow cytometry. Stained cells were read on a Gallios analyzer (Beckman Coulter) and 20,000 events were counted.

Analysis of the cell cycle was performed using the PI/Cell Cycle Analysis Kit (Canvax Biotech, Córdoba, Spain). MCF-7 cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/well and left to grow for 24 h. Afterwards, the cells were incubated in the presence of 0.15 mg·mL⁻¹ or 0.6 mg·mL⁻¹ of SKE, 0.1 mM or 0.4 mM of RA or DMSO (vehicle) for 48 h. The cells were harvested, sedimented by centrifugation (1000 rpm, 5 min), washed with ice cold PBS, centrifuged again in the same conditions and fixed in ice cold 70% ethanol for 45 min. Cells were recovered by centrifugation, washed with PBS, centrifuged again, re-suspended with 200 µL of staining solution containing propidium iodide and RNase A, and incubated at 37 °C for 30 min in the dark. The cells were analyzed by flow cytometry using a Gallios analyzer and Kaluza software (Beckman Coulter, Brea, CA, USA). The percentage of cells in each phase of the cell cycle was calculated and the apoptotic cells were considered to constitute the sub-G0/G1 cell population.

Bacterial strains were grown to OD_600nm = 0.4 (no stress) in BHI or BHI supplemented with 0.5 M NaCl during 30 min (stress). Around 3 × 10⁸ bacteria were spun down by centrifugation (10,000 × g, 18 min, 4 °C) and washed twice in PBS, pH 7.4. The cells were fixed with 4% (w/v) paraformaldehyde for 15 min at room temperature. Fixed cells were harvested by centrifugation and resuspended in 500 µL of filtered PBS, pH 7.4. Quantification of single cell fluorescence was achieved by flow cytometry with Beckman Coulter GALLIOS Analyzer with 488 nm blue laser excitation and 50,000 events recorded for each sample. The data collected were processed with Kaluza software to plot side and forward scatter values, the percentage of eGFP-positive cells and the mean of fluorescence values. Statistical analysis has been carried out by one-way ANOVA (Bonferroni’s multiple comparison test).

Growing fibroblasts were washed with PBS and incubated with 50 nm tetramethylrhodamine, ethyl ester (TMRE) (Molecular Probes) in prewarmed PBS for 30 minutes at 37°C. Cells were trypsinized, centrifuged at 1,000g for 5 minutes, and resuspended in prewarmed PBS. All samples were captured by a Gallios Analyzer (Beckman Coulter), which recorded 20,000 cells for each genotype tested. FCCP (200 μM for 10 minutes) was used as a positive control. Histograms showing the inner Ψm levels were obtained after gating live cells. The data were analyzed with Kaluza Analysis software, version 2.1. (Beckman Coulter).

Cells were plated on 6-well plates and incubated overnight. The test compounds were added. Cells were incubated again overnight and then were harvested for the assay. For the apoptosis assay, the Annexin V-FITC apoptosis detection kit (Cat. No.: APOAF) was purchased from Sigma (Saint Louis, MO). Into 500 μls of cell suspension was added 5 μl of Annexin V-FITC conjugate and 10 μl of a propidium iodide solution. The mixture was incubated for 10 minutes and assayed to determine fluorescence using a Beckman-Coulter Gallios analyzer. Data were analyzed with Gallios software. For the cell cycle assay, the Coulter DNA Prep reagents kit (Cat. No.: PN 6607055) from Beckman Coulter (Fullerton, CA) was used. Analysis was done on a Beckman-Coulter Epics FC500 analyzer using CXP software for acquisition and the ModFit LT v3.1 (Verity Software) for cell cycle modeling. Both experiments were repeated three times.