Eclipse ti e inverted fluorescence microscope

Cells were imaged on a Nikon Eclipse Ti-E inverted fluorescence microscope with a 100X (NA 1.40) oil-immersion objective. Images were collected on a DU897 electron multiplying charged couple device camera (Andor) using µManager v. 1.4 (Edelstein et al., 2010 (link)). Cells were maintained at 30°C during imaging with an active-control environmental chamber (Haison).
Cultures grown for 48 h were diluted 100-fold into PBS and 2 µL were spotted onto a 1% (w/v) agarose MRS pad. After drying at room temperature, the pads were covered with a cover slip, sealed with a mixture of equal portions of Vaseline, lanolin, and paraffin, and transferred to the microscope. Images were taken every 2 min using µManager v. 1.4.
To quantify the morphology of cells using fluorescent strains, co-cultures were diluted 100-fold into PBS and 2 µL were spotted onto a 1% (w/v) agarose PBS pad. After drying, the pads were covered with a cover slip and transferred to the microscope. Images were acquired at room temperature using µManager v. 1.4.

Oli-neu cells were plated on poly-L-lysine-coated Ibidi 8-well chamber slides (BioValley, Marne la Vallee, France) and exposed to BPA. Then, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% triton and saturated with 3.5% donkey serum in PBS. Immunocytofluorescence staining was performed using mouse anti-MBP (1:200, Merck-Millipore MAB382, Guyancourt, France) and mouse anti-CNPase (1:250, Merck-Millipore MAB326, Guyancourt, France). Cells were incubated overnight with primary antibodies at 4 °C, washed three times, and incubated with donkey anti-mouse secondary antibody conjugated to Alexa fluor 488 (1:500, Invitrogen A21202, Villebon-sur-Yvette, France) for 1 h. DAPI was used to counterstain nuclei (Sigma D9542, Saint Quentin Fallavier, France). Images were acquired using an Eclipse Ti-E inverted fluorescence microscope and the NIS-Elements software (Nikon, Champigny sur Marne, France).

To examine LCV architecture and integrity, and enumerate the number of bacteria per LCV, 4x10⁵ macrophages were plated on coverslips in a 24-well tissue culture plate and incubated overnight. L. pneumophila were grown to post-exponential phase then used to challenge macrophages at a MOI = 1 for 1 hr. Cells were rinsed 3 times with culture medium either containing FBS (exogenous lipids) or lacking FBS (no exogenous lipids). At 6 and 12 hpi, cells were fixed in 4% paraformaldehyde in 1x PBS for 30 min at room temperature in the dark. Cells were permeabilized with ice-cold methanol and stained with a chicken α-Legionella antibody (1:5,000) [48 (link)] and rat α-Galectin-3 antibody (Santa Cruz) (1:1,000) in 1x PBS containing 4% goat serum (Gibco) followed by goat α-chicken IgG Alexa Fluor 594 (1:500) (Molecular Probes) and goat α-rat IgG Alexa Fluor 488 (1:1,000) (Molecular Probes). Samples were mounted in SlowFade Gold Antifade reagent (Invitrogen) and imaged with a Nikon Eclipse Ti-E inverted fluorescence microscope in three dimensions acquiring Z stacks at 0.2 μm intervals using NIS Elements software (vAR 5.21.03). Images were deconvoluted and signal intensity, volume and surface area in three-dimensional space were quantified using Volocity (v7.0.0).

BeWo cells were incubated with Calcein AM (Thermo Scientific; C1430; 1:200) and Ethidium homodimer-1 (Thermo Scientific; E1169; 1:200) and imaged using an Eclipse Ti-E Inverted Fluorescence Microscope (Nikon). Green fluorescence indicated live cells and red fluorescence indicated dead cells. Images were analyzed using Fiji (National Institutes of Health) and NIS Elements software (Nikon), and the percentage ratio of live to dead cells were calculated by dividing the mean intensity of live cells (as determined by fluorescence of Calcein AM stain) by the mean intensity of the dead cells (as determined by fluorescence of Ethidium homodimer-1 stain).
Cell proliferation was determined using a CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit (MTS Assay; Promega). The absorbance was measured at 490 nm on a Multiskan® Spectrum spectrophotometer (Thermo Scientific). Given that the absorbance is directly proportional to the number of live cells, relative rate of cell proliferation was determined by calculating the fold change in absorbance compared to the 2D control. Matrigel samples with no cells seeded were used to correct for any potential background absorbance.

Overnight cultures were diluted 1:200 (or 1:100 for ΔbamD cultures due to their low cell density) and grown at 37 °C, followed by two 1:10 dilutions (at OD₆₀₀ around 0.1) to generate steady-state log-phase cells. Cells were imaged on a Nikon Eclipse Ti-E inverted fluorescence microscope with a 100X (NA 1.40) oil-immersion objective (Nikon Instruments). Images were collected on a Zyla 5.5 sCMOS camera (Andor Technology) using µManager v. 1.4^{37 (link)}. Cells were maintained at 37 °C during imaging with an active-control environmental chamber (HaisonTech).
For single-cell imaging on agarose pads, 1 µL of cells was spotted onto a pad of 1% agarose in fresh LB. For microfluidic flow-cell experiments, including the spent medium assays, the oscillatory osmotic shock assays, and the plasmolysis/lysis assays, cells were loaded into CellASIC ONIX microfluidic flow cells (Sigma-Aldrich, Cat. #B04A-03-5PK) and medium was exchanged using the CellASIC ONIX2 microfluidic platform (Sigma-Aldrich, Cat. #CAX2-S0000).

Fluorescence in situ hybridization (FISH) assays were performed using a FISH Kit (RiboBio, Guangzhou, China). BXPC-3 and PANC-1 cells were washed with phosphate-buffered saline (PBS) and fixed in 4% formaldehyde for 10 min. The cells were then permeabilized with PBS containing 0.5% Triton X-100 at 4 °C for 5 min and washed with PBS three times for 5 min, followed by prehybridization at 37 °C for 30 min. The cells were hybridized at 37 °C overnight in the dark with an anti-miR-548t-5p, anti-U6, or anti-18S oligodeoxynucleotide probe, counterstained with DAPI (4′,6-diamidino-2-phenylindole) and imaged using a Nikon Eclipse Ti-E inverted fluorescence microscope (Nikon, Japan).

Cell-hydrogel constructs were fixed in 4.0 v/v % paraformaldehyde (Sigma-Aldrich) in PBS for 30 min at room temperature, blocked and permeabilized in 0.2 v/v % Triton X-100 (Sigma-Aldrich) and 5 v/v % bovine serum albumin (Sigma-Aldrich) in PBS for 1–2 h at room temperature. Each step described above was followed by three washes with PBS for 5 min. The constructs were then incubated for overnight at 4 °C in 0.2 v/v % Triton X-100 and 0.2 v/v % bovine serum albumin (Sigma-Aldrich) in PBS with primary antibodies, Nestin (1:500; Millipore), GFAP (1:1000; Sigma-Aldrich), Neurofilament (1:1000; Abcam, UK), and βIII-tubulin (1:1000; Sigma-Aldrich) (Table S2). After a minimum of 3 washes with PBS for 1 h on the shaker at low speed, the constructs were incubated for overnight at 4 °C in 0.2 v/v % Triton X-100 and 0.2 v/v % bovine serum albumin in PBS with NucBlue Live ReadyProbes™ Reagent (Invitrogen) or Hoechst 33342 staining solution (Thermo Fisher Scientific), and Alexa Fluor secondary antibodies (Thermo Fisher Scientific), followed with 3 washes with PBS for 1 h on the shaker at low speed. The stained samples were stored at 4 °C and the images were acquired with a Nikon Eclipse Ti-E inverted fluorescence microscope (Nikon Instruments Inc., UK).