Axio observer z1

Aggregation phenotypes were directly observed after cell resuspension in TBS buffer (pH 7.4) or TBS buffer supplemented with 1 mM ZnCl₂, addition of 1 mM EDTA, and further addition of 1 mM ZnCl₂. Aggregation levels were observed in test tubes, by optical microscopy at low magnification (Zeiss Stemi DV4 stereomicroscope; Oberkochen, Germany) and at high magnification (Zeiss Axio Observer Z1 equipped with a Hamamatsu C10600 camera; Oberkochen, Germany).

Undifferentiated and differentiated 3T3-L1 cells were grown on coverslips coated with collagen IV (Sigma-Aldrich). Serum-starved cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 10 min and permeabilized with 0.2% Triton X-100 for 3 min at room temperature. After blocking with 5% donkey serum for 1 h, cells were stained overnight at 4°C with primary antibodies, followed by incubation with secondary antibody for 1 h at room temperature. In several experiments, cells before fixing were incubated with LysoTracker (0.4 μM) for 40 min at 37°C. Antifade solution was used for mounting cells on slides. Slides were examined with the help of the Axio Observer Z1 fluorescence microscope equipped with the Hamamatsu digital camera C10600/ORCA-R2 and AxioVision 4.8.1 program (Carl Zeiss, Thornwood, NY). Each scale bar is 5 μm. Each image shows a representative result of at least three independent experiments.

To assess the adhesion phenotype of the bacterial strains, bacteria were incubated with Fn-coated substrates prepared as follows. Glass coverslips coated with a thin layer of gold were immersed overnight in an ethanol solution containing 1 mM 10% 16-mercaptododecahexanoic acid–90% 1-mercapto-1-undecanol (Sigma), rinsed with ethanol, and dried with N₂. Substrates were then immersed for 30 min in a solution containing 10 mg ⋅ ml⁻¹N-hydroxysuccinimide (NHS) and 25 mg ⋅ ml⁻¹ 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (Sigma), rinsed 5 times with Ultrapure water (ELGA LabWater), incubated with 0.1 mg ⋅ ml⁻¹ of Fn from bovine plasma (Sigma) for 1 h, and rinsed further with phosphate-buffered saline (PBS) buffer. Fn substrates were incubated at 37°C in 200-µl bacterial suspensions adjusted in Tris-buffered saline (TBS) buffer supplemented with 1 mM ZnCl₂ to an OD₆₀₀ of 0.3 to 0.4. After 2 h, the substrates were gently rinsed by 3 consecutive washes in TBS buffer supplemented with 1 mM ZnCl₂ and directly imaged using an inverted optical microscope (Zeiss Axio Observer Z1) equipped with a Hamamatsu C10600 camera.