35 mm petri dishes

Fresh whole human blood (French National Blood Service) was centrifuged (2,000 × g, 10 min, 4°C). Three components were obtained at this stage: (i) the upper phase, a clear solution of blood plasma; (ii) a middle thin layer of platelets and leukocytes; and (iii) at the bottom RBCs. RBCs were collected and washed twice in cold 1× Dulbecco’s PBS (DPBS; Gibco). An equivalent MOI of 40 bacteria was incubated with 2 ml of RBC suspension (≈10,000 RBCs) in 35-mm petri dishes (MatTek) at 37°C. Bacterial overnight cultures were directly added to the petri dishes during microscopy acquisition. Live-cell imaging was performed during 60 min on a Zeiss Axio Observer spinning-disk confocal microscope equipped with a 63×oil objective and driven by the MetaMorph software. Images were acquired every 5 s for 60 min. One hundred RBCs were counted for hemolysis for each of the strains tested during the duration of the experiment. Three independent experiments were performed.

To detect the expression of myc-pBCL-G and HA-pJAB1, SUVEC cells were seeded in 35-mm petri dishes (MatTek, MA, USA) and were co-transfected with pCMV-myc-pBCL-G and pCMV-HA-pJAB1 vectors. About 24 hours after transfection, cells were fixed with 4 % paraformaldehyde for 30 minutes at room temperature and permeabilized with 0.2 % Triton X-100/PBS for 5 minutes. Then, cells were incubated with mouse anti-myc tag (Sigma Aldrich, Saint Louis, USA) or anti-HA tag (Sigma Aldrich, Saint Louis, USA) monoclonal antibody (1:1000 dilution) for 1 hour at room temperature, followed by incubation with FITC-conjugated goat anti-mouse IgG secondary antibody (Sigma Aldrich, Saint Louis, USA, 1:100 dilution) for 1 hour at room temperature. After being rinsed with PBS, cells were observed using an inverted fluorescence microscope.

Cells were plated in glass bottom 35 mm-petri dishes for 24 h (No. 1.5, MatTeK Corporation, United States) and transiently transfected with the fluorescence resonance energy transfer (FRET)-based D1ER cameleon (Palmer et al., 2004 (link)). FRET imaging was described as in the previous report (Chen et al., 2019 (link)). Briefly, cells were imaged by an Epi-fluorescence microscope with a Plan-Fluor 40×/1.3 Oil objective (Eclipse Ti, Nikon, Japan). The emission ratio of the cameleon was accomplished by 425 nm excitation wavelength with a dichroic mirror 515 nm and two emission filters (475 nm for ECFP and 535 nm for citrine-YFP) (Chroma Technology Corporation, United States). Changes in ER Ca²⁺ were expressed as the FRET-to-CFP emission ratio. Images were analyzed using MetaFluor software (Universal Imaging Corporation, United States).