Propidium iodide

Prior to transplantation, encapsulated islets were stained for viability. Cells were incubated in calcein AM (4 μM, ThermoFisher) and propidium iodide (1 μg/mL, ThermoFisher) for 30 minutes. Fluorescence was captured with a Cytation 5 Imaging Multi-Mode Reader (Biotek Instruments). Using the Cytation software, the area of calcein-stained (live) cells was divided by the total cell area (obtained with brightfield images of the same fields) resulting in the percentage of live cells. Following encapsulation, microspheres were incubated in calcein AM (4 μM, ThermoFisher) and propidium iodide (1 μg/mL, ThermoFisher) for 40 minutes and the same procedures followed as described for unencapsulated cells.

Cells were seeded at a density of 2 × 10⁵ cells/well in DMEM medium in six-well plates. The next day, cells were incubated with the indicated treatments. After that, the cells were then stained with propidium iodide (Thermo Fisher Scientific, R37108) for 30 min in an incubator of 5% CO₂ at 37 °C. Morphological changes were examined by fluorescence microscope at ×20 magnification. A Countess II FL Automated Cell Counter (Thermo Fisher Scientific) was used to assay the percentages of dead cells after propidium iodide staining.

For each condition, BS were distributed onto a Thermanox^TM coverslip (cell culture treated side up) placed at the bottom of a 24 well plate (500 µL per well). After 24 h of incubation, the coverslips were washed twice in PBS and stained with SYTO^TM 9 at 1 µM to label bacteria and (i) propidium iodide (Thermo Fisher Scientific, USA) at 20 µM (in this case to label live bacteria with SYTO^TM 9 and damaged or “dead” bacteria with propidium iodide); (ii) SYPRO^® Ruby (v/v) to label proteins; or (iii) wheat germ agglutinin (WGA) associated with the Alexa Fluor^TM 350 conjugate at 100 mg/mL to label PIA and TOTO^TM-3 iodide at 2 mM to label extracellular DNAs (all from Thermo Fisher Scientific, USA). Each label was diluted in 0.9% NaCl. After 30 min of incubation in the dark at room temperature, each coverslip was washed two times with PBS and placed in a 24 well Krystal plate with glass bottom (Porvair, Whiteley, UK) with the biofilm-side downward facing. Acquisitions were performed using CLSM (LSM 710 NLO, Zeiss, Oberkochen, Germany). Fluorochrome-labelled compounds were imaged, and their volume quantified using IMARIS software (v. 9.8.0). Two biological replicates were used for acquisitions. For each coverslip, three representative acquisitions were done.

HCT-116^p53+/+ and HCT-116^p53−/− cells were treated either with DMSO as a negative control or with 15 μM XCT790 for 48 h. Cells were then harvested and incubated with 1x Annexin V binding buffer containing Annexin V-FITC (BD Biosciences, Franklin Lake, NJ, USA) and propidium iodide (PI, Thermo Scientific) for 15 min in the dark at room temperature. Fractions of apoptotic cells were measured using flow cytometry. Also, for accessing the cell cycle progression of HCT-116^p53+/+, HCT-116^p53−/−, and DLD-1 cell lines, cells were harvested and incubated with propidium iodide (PI, Thermo Scientific) and RNase for 30 min in the dark at room temperature to measure cellular DNA content. All analyses were performed on LSRFortessa X-20 flow cytometer (San Jose, CA, USA) and the BD FACSDiva Software version 8.0.2. Data were analyzed with the BD FlowJo version 10 software.