Celltracker orange cmra

To image 3D microtissue assembly and quantitate volumetrics, cells were stained with either CellTracker Deep Red, CellTracker Green CMFDA, or CellTracker Orange CMRA (Molecular Probes/Thermo Fisher Scientific) using the manufacturer’s protocol prior to assembly into microtissues. The labeled cells were imaged in live microtissues using confocal fluorescence microscopy. For volumetric analysis, live microtissues were imaged using quantitative high content imaging (Opera Phenix, Perkin Elmer) and Harmony analysis software was used to identify each fluorescently-labeled cell population. Z-stacks of intact microtissues were obtained using confocal laser scanning fluorescence microscopy throughout the depth of the microtissue. Microtissue volume was calculated based on analysis of 142, 177, and 96 microtissues on days 2, 4, 7, respectively.

Mammalian cells were incubated for 20 min in a 10 µM solution of CellTracker Orange CMRA (Invitrogen, C34551) and washed with DPBS before seeding the bacteria. For standard visualization of biofilm grown on top of epithelial monolayers, since V. cholerae strains were not constitutively fluorescent, samples were incubated for 20 min with a 10 µM solution of SYTO9 (Invitrogen, S34854) and washed with DPBS before visualization. This results in double staining of epithelial cells. For the visualization of live and dead cells in infected monolayers we instead incubated the samples for 20 min in a solution containing 5 µg/ml Hoechst (Thermo Fischer Scientific, 62249) and 5 µM Calcein-AM (Sigma Aldrich, 17783). For the visualization of epithelial cells monolayers permeability, we added 1 ml of a 2 µM solution of fluorescein isothiocyanate-dextran (Sigma Aldrich, 46944) on top of the cells and imaged after 30 min.
V. cholerae biofilms grown in microfluidic channels were incubated for 20 min with a 10 µM solution of SYTO9 (Invitrogen, S34854) and washed with M9 minimal medium before visualization.

B16F10 cells were stained with cell tracker orange CMRA (Invitrogen) and viral-transduced OTI CD8⁺ T cells were incubated with cancer cells in the presence or absence of OVA peptide (257–264, 1 μg/ml) at a 5:1 ratio in 5 ml round-bottom polystyrene tubes at 37°C for 4 h. After incubation, 10 μL of a 5 μg/ml solution of 7-AAD was added to the cell suspension for 10 min at RT. The cells were evaluated on a FACS Canto (BD Biosciences), and the data were analyzed with FlowJo software (TreeStar).

T cells were labeled with CellTracker Orange CMRA (Invitrogen, C34551) for transmigration experiments as previously reported (De Haan et al., 2021 (link)). Briefly, T cells were incubated with 2.5 μM CellTracker Orange CMRA for 30 min. Labeling was terminated by adding AIM-V medium. The T cell suspension was centrifuged and resuspended in fresh AIM-V medium at a final density of 400,000 cells/mL. For transmigration assays, 20,000 T cells were perfused through the lumen of the endothelial vessel (top lane) followed by addition of 800 ng/mL CXCL12 (Peprotech, 300-28A) to the opposite (bottom) lane. The OrganoPlate was placed on the OrganoFlow rocker (7° inclination, 8 min interval) in the incubator (37°C, 5% CO₂) and migration was tracked at 0, 24 and 48 h after T cell addition using the ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices). For blocking experiments, T cells were pre-incubated with 50 μg/mL anti-VLA4 antibody Natalizumab (MedChem Express, HY-108831) for 45–60 min before addition to the microfluidic chips. The same concentration of Natalizumab was added to the lumen of the HBMEC vessels.

ASK cells were seeded into a 24 well-plate at a density of 1 × 10⁵ cells/well. After 2 days, the cells were incubated for 40 min with the fluorescent probe CellTracker™ Orange CMRA (Invitrogen, USA) at a final concentration of 10 μM. Then, the cells were washed three times with PBS and treated with attenuated E. coli HT115 (carrying the green pBADT plasmid) overexpressing green fluorescent protein (GFP) in supplemented L-15 media at a multiplicity of infection (MOI) of 500 bacteria/cell (the maximum MOI that did not cause cell damage). Attenuation of the bacterial culture was performed by adding formaldehyde at a final concentration of 0.5% v/v and incubating for 20 min at room temperature. Immediately after this step, the bacterial culture was centrifuged and washed twice with PBS, quantified in a Petroff-Hausser chamber and used for analysis. The incubation of ASK cells with attenuated bacteria in supplemented L-15 media was followed by 4 days at 15°C. During the assay, slides with cell samples were removed at 0, 24, 48, and 96 h. Each slide was fixed with 4% paraformaldehyde (PFA) for 10 min and then treated with 50 mM NH₄Cl for 10 min. Finally, the slides were mounted in 1,4-diazabicyclo[2.2.2]octane (DABCO) and visualized using a Nikon C2⁺ confocal microscope. Phase contrast photographs were taken using a Carl Zeiss LSM 510 confocal microscope with a DIC 3.4 filter.