Hoechst 33342

HeLa cells in the logarithmic growth phase were trypsinized, washed twice with PBS, and counted. Then, the HeLa cells were resuspended in DMEM with 2% FBS (5 × 10⁶ cells/mL) and divided into two groups. Group 1 was incubated with the DNA binding dye Hoechst 33342 (Sigma Aldrich, USA) at a final concentration of 5 ug/mL for 90 min at 37°C with gentle agitation every 15 min. Group 2 was pretreated with 50 μg/mL verapamil (Sigma Aldrich, USA) for 15 min at 37°C, and then incubated with Hoechst 33342 (final concentration: 5 μg/mL) for 90 min at 37°C with gentle agitation every 15 min. The incubation process was carried out in the dark. The cells were then washed twice with ice-cold PBS and resuspended in PBS containing 2% FBS and 10 mM HEPES. The cell suspension was stored at 4°C while protected from light before FACS. Cell suspensions were freshly prepared for cell sorting and stained with PI (Sigma Aldrich, USA) at a final concentration of 2 μg/mL. Cell sorting was performed using a FACS DIVA fluorescence-activated cell sorter (BD Biosciences, USA). SP and non-SP cells were collected separately in sterile 25-cm² flasks and cultured in DMEM containing 10% FBS at 37°C with 5% CO₂. Cell morphology was examined under an inverted microscope every 6 h.

Apoptosis of NPCs was assessed by examining nuclear morphology in Hoechst 33342-stained cells as described previously.^{67 (link)} Briefly, NPCs were stained with 1 μg/ml Hoechst 33342 (Sigma) and the fraction of cells exhibiting an apoptotic nuclear morphology characterized by chromatin condensation and/or apoptotic bodies was quantified. In certain experiments, NPC death was determined by Live/Dead assay according to the manufacturer's instructions (Invitrogen). Briefly, NPCs were stained with Calcein-AM (2 μM) and ethidium homodimer (4 μM) for 20 min and the fraction of live (Calcein-AM-positive) and dead (ethidium-positive) cells was scored. NPCs were visualized by fluorescence microscopy (Zeiss, Toronto, ON, Canada) and images were captured with a Zeiss Axio-Cam camera (Zeiss). Images were captured and scored by an observer blinded to the treatment. A minimum of 500 cells from five randomly selected fields were analyzed for each treatment and the fraction of apoptotic or dead cells was determined.

Hoechst 33342 exclusion (side population) assays were carried out as previously described [30] to analyse cells overexpressing ABC transporters. Single‐cell suspension obtained from parental cell lines and melanospheres were stained with Hoechst 33342 (Sigma‐Aldrich) dye. As negative controls, verapamil (Sigma‐Aldrich) was used for maintaining the efflux channel closed, inhibiting the capacity to efflux Hoechst 33342 by cells. The bright fluorescent cells were measured by flow cytometry in Hoechst blue (440/40) and Hoechst red (695/40) of a FACScan Aria III (BD Biosciences) using FACSDIVA software from the CIC Scientific Instrumental Centre (University of Granada). Cells with the ability to efflux Hoechst 33342 were considered as the side population (SP).

To evaluate the percentage of side population, the cells were stained with Hoechst 33,342 (Sigma-Aldrich) as described in [34 (link)]. Briefly, 1 × 10⁶ cells were stained with 20 μg/mL Hoechst 33,342 for 90 min and 50 μM verapamil (Sigma-Aldrich) was treated to establish the population. After washing with PBS, we analyzed the population using flow cytometry at 355 UV light to detect the fluorescence with 450/20 band-pass filter and red fluorescence with a 675/20 filter.

HeLa cells were seeded onto 6-well plates containing sterile coverslips at a density of 7 × 10⁴ cell/mL. Following infections with V. parahaemolyticus strains at an MOI of 10 and addition of CNF1/CNF1 C866A as detailed above, the cells were washed with PBS and fixed in 3.2% (vol/vol) paraformaldehyde for 10 min at room temperature. Fixed cells were washed in PBS and permeabilized with 0.1% Triton X-100 for 10 min at room temperature. Nuclei and actin cytoskeleton were stained with Hoechst 33342 (Sigma) and rhodamine-phalloidin (Molecular Probes), respectively, for infection analyses and quantification. For evaluating endosomal localization of bacteria, nuclei, actin cytoskeleton, and LAMP-1 were stained with Hoechst 33342 (Sigma), Alexa Fluor 680-phalloidin (Molecular Probes), and mouse anti-LAMP-1 (Abcam Ab25630), respectively, as described previously (31 (link)). The images were collected using a Zeiss LSM 710 confocal microscope.