Facsdiva

In order to analyse the total amount of infected cells in the LTI macrophages, LTI cells were scraped and resuspended in PBS with 1% BSA (Albumin bovine serum, Sigma, Germany). Cells were counted and 6 × 10⁶ cells ml⁻¹ were analysed using a LSRII (BD Bioscience, CA, USA). Data obtained were evaluated using FACS DiVA (BD Bioscience, CA, USA) or FlowJo Mac v8.84 (Tree Star, Ashland, OR, USA). To analyse cell death, LTI or RI macrophages were scraped and resuspended in PBS with 1% BSA (Albumin bovine serum, Sigma, Germany). Cells were counted and 5 × 10⁶ cells ml⁻¹ were resuspended in binding buffer (0.01 M Hepes pH 7.4, 0.14 M NaCl, 0.25 mM CaCl₂). Cells were stained with APC Annexin V (BD Biosciences, CA, USA) and 7-AAD (7-amino-actinomycin D, BD Biosciences, CA, USA) following the manufacturer's instructions, to distinguish apoptotic and dead/necrotic cells. After staining, cells were stored on ice until analysis. As a control for apoptotic cell death, uninfected RAW 264.7 cells were exposed to UV light for 15 min and incubated for 6 h before analysis. Samples were analysed using a LSRII (BD Bioscience, CA, USA) and data obtained were evaluated using FACS DiVA (BD Bioscience, CA, USA) or FlowJo Mac v8.84 (Tree Star, Ashland, OR, USA).

In the early phase of apoptotic death occurring via the intrinsic pathway, there is an increase in mitochondrial membrane permeability, resulting in a decrease in mitochondrial membrane potential (MMP, ΔΨ_m). To detect these changes, the carbocyanine lipophilic cationic fluorochrome JC-1 included in the JC-1 MitoScreen kit (BD Pharmigen, San Diego, CA, USA) and a flow cytometer (BD FACSCanto II) with the appropriate software to analyze the obtained results (FACSDiva; both from BD Biosciences Systems) were used. The entire cell staining and cytometric analysis procedure was performed according to the instructions provided with the kit. MCF-7 and MDA-MB-231 cells were incubated for 24 h (37 °C, 5% CO₂, 90–95% humidity) with cisplatin and compound EDA-71 (concentrations of 1.5 and 3 µM). After the incubation time with the tested compounds, the cells (1 × 10⁶ cells/sample) were washed and resuspended in 0.5 mL of buffer containing 10 µg/mL JC-1 dye. Incubation was carried out for 15 min at room temperature, protected from light. Afterwards, the cells were washed twice with buffer, resuspended in 0.5 mL PBS and immediately analyzed using a flow cytometer (BD FACSCanto II; 10,000 events measured) and FACSDiva software to count the percentage of cells with reduced ΔΨ_m. The equipment was calibrated with BD Cytometer Setup and Tracking Beads (BD Biosciences, San Diego, CA, USA).

CAR-modified immune cells were stained with fluorescence-conjugated antibodies in FACS staining buffer (PBS with 1% FBS) on ice for 30 min on ice in the dark. Then, cells were washed with PBS, and analyzed on either a FACS LSRII or an LSR Fortessa flow cytometer (BD). Photomultiplier tube voltages were adjusted using the FACS Diva (BD) software and compensation values were calculated before data collection. Flow data were then acquired using FACS Diva (BD) and analyzed using FlowJo (Tree Star).
For flow cytometry single live cell sorting, sample cells were stained with fluorescence-conjugated antibodies with (RPMI-1640 with 1% FBS) on ice for 30 min, washed with PBS twice, resuspended in completed culture medium, and sorted by SORP BD FACS Aria III. After sorting, collection samples were washed with prewarmed medium once, and cultured for use.

HEK293 cell lines containing a stably integrated copy of the EJ5-GFP reporter were used to measure the repair of I-SceI-induced DSBs by NHEJ (Bennardo et al. 2008 (link)). Briefly, 48 h after siRNA transfection, cells were cotransfected with a mCherry expression vector and the I-SceI expression vector pCBASce. Forty-eight hours later, the percentage of GFP-positive cells among mCherry-positive cells was determined by FACS on a BD LSRII flow cytometer (BD Bioscience) using FACSDiva software version 5.0.3. Quantifications were performed using WinMDI 2.9 (freeware), FACSDiva (BD Biosciences), or FlowJo software (Flowing Software 5.2.1.).

Cell cycle analysis was carried out by flow cytometry. Briefly, MCF10A cells were seeded into 6-well culture plates, treated as indicated, collected, fixed, stained with propidium iodide (100 µg/mL) and RNAse (20 µg/mL) in PBS for 1 h, acquired on a FACSCanto II flow cytometer instrument and analyzed using FACSDiva and FlowJo software (BD Biosciences). Similarly, indirect immunofluorescence on ethanol-fixed MCF10A cells (70% in PBS) was performed to quantify mitotic cells using an anti-phospho-H3 (Ser10) specific antibody (CellSignaling) detected by an Alexa488-labeled goat anti-rabbit secondary antibody (Invitrogen). Cells were acquired on a FACSCanto II flow cytometer instrument and analyzed using FACSDiva and FlowJo software (BD Biosciences). Ten thousand events were analyzed for each sample.

Initially, 24-well plates were coated with ECM of fibronectin and laminin, respectively (Sigma–Aldrich, St. Louis, MO, United States) at a concentration of 50 μg/mL for 18 h at 4°C followed by incubation for 1 h at 37°C. The plates were then washed three times with PBS and fungal suspension was added at a concentration of 1.5 × 10³ CFU/mL, which had been inoculated into the 24-well plate previously sensitized with ECM and incubated at 37°C for 1, 3, or 5 h. Subsequently, the supernatant was removed, the plate was washed with PBS, and then 300 μL of trypsin was added (Sigma). The supernatant was obtained and then centrifuged at 10,000 rpm for 5 min at (4°C). The new supernatant was discarded and 500 μL of FACS buffer was added for subsequent cell counting by flow cytometry (BD FACS Canto and BD FACS Diva software analysis). Following the adhesion period, treatment with the selected compound (dodecyl and NLS+dodecyl) was performed for 24 h at 37°C. RPMI-1640 with L-glutamine and 2% glucose was used to maintain adequate nutritional status. Subsequently, the supernatant was removed and the plate was washed with PBS. Then, 300 μL of trypsin was added and centrifuged. The supernatant was discarded and 500 μL of FACS buffer was added to each tube for subsequent cell counting by flow cytometry (BD FACS Canto and BD FACS Diva software analysis).