Caco-2 cells were grown for 5–7 days in an 8-well chamber slide (μ-Slide 8 well Glass bottom, Ibidi). Then, cells were stimulated with COF-SN (0.5 mg/ml) or OMVs (0.1 mg/ml) for 24 h at 37°C. After washing with PBS cells were fixed with 3% paraformaldehyde in PBS, permeabilized with 0.05% saponin (Sigma–Aldrich) and blocked using PBS containing 1% bovine serum albumin. The TJ proteins ZO-1, claudin-2 and occludin were stained using, respectively, anti-ZO-1 (5 μg/ml, Invitrogen), anti-claudin-2 (5 μg/ml, Abcam) rabbit IgG antibodies, and anti-occludin mouse IgG antibody (0.5 μg/ml, Invitrogen) for 16 h at 4°C, followed by incubation with Alexa Fluor 488-conjugated F(ab’)2 goat anti-rabbit IgG (H+L) (5 μg/ml, Invitrogen) or Alexa Fluor 488 - conjugated F(ab’)2 goat anti-mouse IgG (H+L) (5 μg/ml, Invitrogen) for 2 h at 37°C. Nuclei were labeled with DAPI (0,125 μg/ml, Sigma–Aldrich) for 20 min at room temperature.
μ slide 8 well glass bottom
Manufactured by Ibidi
Sourced in Germany
The μ-Slide 8 Well Glass Bottom is a multi-well cell culture slide with a glass bottom. It is designed for high-resolution microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Matlab, by MathWorks (2 mentions)
Deltavision elite system, by Cytiva (2 mentions)
Lsm 800 microscope, by Zeiss (2 mentions)
Anti zo 1, by Thermo Fisher Scientific (1 mentions)
Anti claudin 2, by Abcam (1 mentions)
Anti occludin mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Saponin, by Merck Group (1 mentions)
Stage top incubator, by Okolab (1 mentions)
Ixon 897 emccd camera, by Oxford Instruments (1 mentions)
1.49 na tirf objective, by Olympus (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Zen black software, by Zeiss (1 mentions)
Alexa fluor 594 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Alexafluor 647 conjugated donkey anti goat antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (1 mentions)
Cellmask orange, by Thermo Fisher Scientific (1 mentions)
18 protocols using μ slide 8 well glass bottom
1
Immunofluorescent Analysis of Tight Junction Proteins
2
Biofilm Formation Assay in Microplates
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Biofilm formation in polystyrene microtitre plates was tested using crystal violet staining assay50 (link). Briefly, overnight cultures of samples were diluted in TB to OD600 0.05, and 300 μl of each sample was added into the wells of 96-well plate (Corning Costar, flat bottom; Sigma-Aldrich, Germany). After 24 h of incubation at 37 °C, the OD600 of the samples was measured, the wells were rinsed with H2O and 300 μl of 1% crystal violet solution was added to each well. After 15 min incubation at room temperature, the wells were rinsed three times with H2O. Remaining crystal violet was solubilized by adding 300 μl of 96% ethanol, and the OD595 of the solution was measured. Values of crystal violet staining were normalized for each sample by the respective OD600.
For biofilm imaging, overnight cultures carrying a high-copy number plasmid pVS1515 encoding egfp were diluted in TB to OD600 0.05 and grown with shaking at 30 °C or 37 °C to the mid-exponential phase (OD600=0.6). The samples were then once again diluted in fresh TB containing 5 μM IPTG to OD600=0.05, and 350 μl of each sample was loaded into the wells of 8-well glass bottom slides (μ-Slide, 8-well glass bottom; ibidi). The cultures were grown at 30 and 37 °C for 24 h without shaking.
For biofilm imaging, overnight cultures carrying a high-copy number plasmid pVS1515 encoding egfp were diluted in TB to OD600 0.05 and grown with shaking at 30 °C or 37 °C to the mid-exponential phase (OD600=0.6). The samples were then once again diluted in fresh TB containing 5 μM IPTG to OD600=0.05, and 350 μl of each sample was loaded into the wells of 8-well glass bottom slides (μ-Slide, 8-well glass bottom; ibidi). The cultures were grown at 30 and 37 °C for 24 h without shaking.
3
TIRF Microscopy of NPY-mRFP and LifeAct-GFP
4
Generation of Bone Marrow-Derived Macrophages
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Bone marrow-derived macrophages were prepared from bone marrow cells harvested from femoral, tibial, and pelvic bones. Cells were cultured on 15-cm non-treated dishes for 6 days (37°C, 10% CO2) in 25 mL Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS, Sigma), 50 U/mL penicillin and 50 μg/mL streptomycin and supplemented with 15%–20% L929 cell-conditioned medium. An additional 10 mL culture medium was added on day 3. Unless otherwise specified in the figure legends, differentiated BMDMs were harvested and replated in sterile 24-well non-treated tissue culture plates at 6x105 cells/well in a final volume of 600 μL DMEM/FCS supplemented with 20% L929 conditioned medium. BMDMs that were to be stimulated with compound A were plated at 4x105 cells/well in a final volume of 600 μL DMEM/FCS supplemented with 20% L929 conditioned medium. For imaging experiments, 2x105 BMDMs were seeded into μ-Slide 8 Well Glass Bottom (Ibidi, 80827) in 200 μL DMEM/FCS supplemented with 20% L929 conditioned medium.
5
Monitoring Lipid Droplets Over Time
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To monitor lipid droplets at the same location over time, we engraved an “x” into the bottom of a glass sample carrier (μ-slide 8-well glass bottom, ibidi®, Munich, Germany) using a diamond knife (Figure 1 A). The carrier was then plasma cleaned (1 min) to remove organic contaminations from the glass and to prevent absorption of oil droplets to the surface. After placing the samples in the carrier, a lid was glued onto it using an epoxy resin to prevent evaporation. The carrier was then placed on a confocal laser scanning microscope (CLSM, Leica SP8, Wetzlar, Germany) such that the same position can be revisited on consecutive days.
6
Live-Cell Imaging of Mitotic Regulators
7
Astrocyte Calcium Imaging with Mechanical Stimulation
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2‐week old differentiated astrocytes were dissociated into single cells using Accutase and plated on μ‐Slide 8 Well Glass Bottom (ibidi) chambers coated with Matrigel (1:80 diluted) at a density of 1.5 × 105 cells per well. Fourdays later, astrocytes were loaded with fluo‐4 acetoxymethyl ester (Fluor‐4 AM) (Life Technologies) diluted in Neurobasal® Medium (Invitrogen) for 1 hour at 37°C. Astrocytes were then washed for three times and left in Neurobasal® Medium for 30 min at 37°C. A negative control was set up by applying 50 μM 2‐APB (Calbiochem), an inhibitor of the IP3‐dependent calcium release, at this stage. The medium was switched to Dulbecco's Phosphate‐Buffered Saline (Life Technologies) prior to imaging. Glass beads (200 μm diameter) were dropped on top of astrocyte cultures as a mechanical stimulus. Time‐lapse imaging was performed using an Axio Observer.Z1 (Carl Zeiss) epifluorescence microscope at 10× magnification with a 488 nm excitation filter at 37°C and 5% CO2.
8
Dual-Species Biofilm Quantification
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For two-color labeling, overnight cultures of E. coli carrying plasmid pOB2 carrying mCherry under the control of the isopropyl-β-d -thiogalactopyranoside (IPTG)-inducible trc promoter and E. faecalis carrying egfp under the control of the constitutively expressed rplL promoter were diluted in TB containing 5 μM IPTG, to a final OD600 of 0.03. For single-color labeling of E. coli , plasmid pVS1515 carrying egfp under the control of the trc promoter was used. For dual-species biofilm cultivation, the same amounts of E. coli and E. faecalis cells were coinoculated, resulting in a final OD600 of 0.06; 400 μl of each sample was cultivated for 24 h at 37°C in 8-well glass-bottom slides (μ-Slide, 8-well glass bottom; ibidi). The biofilms were visualized using a Zeiss LSM-800 microscope (apochromat 40× objective), and z-stack images were acquired and analyzed using ZEN Black software (Zeiss).
Three-dimensional structures of matureE. coli biofilms (green fluorescent protein [GFP] positive) were quantified using the 3D objects counter plugin for ImageJ (53 (link)). The plugin allows quantification of the volumes of patches formed by connected fluorescent cells.
Three-dimensional structures of mature
9
Imaging Actin and LYVE-1 in Primary HDLECs
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Monolayers of primary HDLECs grown on 0.1% gelatin-coated glass slides (Ibidi μ-Slide 8-well glass bottom (#80827, Ibidi) were rinsed with PBS and fixed in 1% (w/v) paraformaldehyde for 15 min at room temperature (RT). Cells were then rinsed with excess PBS and permeabilized for 1 min in wash buffer (PBS, 10% fetal calf serum, and 0.5% sodium azide) containing 0.1% Triton X-100, prior to the addition of Abberior® STAR 635–labeled phalloidin (#30972-20UG, Sigma–Aldrich, 1:100 final dilution) and mouse anti-human LYVE-1 (mAb 8C, 10 μg/ml) and incubation for 15 min at RT. After rinsing with PBS, a secondary Alexa Fluor 594 goat anti-mouse IgG (#A11005, Thermo Scientific) was added to detect LYVE-1. The cells were then rinsed with PBS and placed in Leibovitz's (L-15) phenol red–free medium (#21083027, Thermo Scientific) for confocal and STED imaging. The images acquired were viewed in Fiji/ImageJ (RSB, National Institutes of Health) (59 (link)), and the CoLoc2 plugin was used to determine the Pearson's correlation between actin and LYVE-1. For labeling actin alone (Fig. S1F ), phalloidin Oregon Green at 1:100 dilution was used as above.
10
Visualizing Bacterial Infection of Live Cells
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Live H292 cells grown on a μ-slide 8 well glass bottom (Ibidi, Munich, Germany) and were infected with biofilm and planktonic bacteria of GFP-expressing GAS strains (ΔhasA or ΔhasAΔslo) using an MOI of 10. The media was removed, cells were washed in PBS (1X) and fixed by 4% PFA as described above. Extracellular bacteria were stained using a primary goat anti-GAS antibody (3.2 µg/ml) diluted 1:500 in 0.05% Bovine serum albumin (BSA) for 30 min. Excess staining was removed by washing in PBS (1X) three times. Secondary AlexaFluor 647 conjugated donkey anti-goat antibody (Invitrogen™, Thermo Fischer Scientific) diluted 1:500 in PBS (1X) was then added for 30 min in the dark. Excess antibody was removed by washing in PBS (1X) three times. Subsequently, cells were permeabilized using Triton X (0.01%) for 15 min at 37°C, washed, and stained using AlexaFluor 568 phalloidin (Invitrogen) using a 1:500 dilution in PBS (1X). DNA was stained by using Hoechst (Thermo Fischer Scientific) diluted to a working concentration of 1 µM in PBS for 30 min and washed for in PBS prior to imaging.
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