Biozero bz 8000

To assess the migratory activity of eEND2 cells, a 24-well chemotaxis chamber and polyethylene therephthalate (PET) filters with 8μm pore size (BD Biosciences) were used. eEND2 cells were exposed to 100, 200 or 400μM geraniol (n = 4 each) or vehicle (DMSO; control; n = 4) for 24h. For the analysis of cell migration, 500μL of a cell suspension, containing 1 x 10⁵ geraniol-treated or vehicle-treated cells in DMEM was added to each of the upper wells. The lower wells were filled with 750μL DMEM supplemented with 1% FCS. The chamber was then incubated for 6h at 37°C in a humidified atmosphere with 5% CO₂. After the 6h incubation period, non-migrated cells were quantitatively removed from the upper surface of the filters. Migrated cells, which were adherent to the lower surface, were fixed with methanol and stained with Dade Diff-Quick (Dade Diagnostika GmbH, München, Germany). The number of these migrated cells was counted in 20 microscopic ROIs (size: 0.15mm²) at 20x magnification (Biozero BZ-8000; Keyence) and is given as cells/ROI.

The ability of hDFSCs to migrate was assessed under aerobic and anaerobic conditions (37 °C). Therefore, a wound closure assay described by Laheij et al.57 (link) was modified. Cells were seeded into 24 well plates with cover slips. The adherent cell layer was continuously scratched, medium was exchanged and cells were infected with 10⁷ cfu/ml of each bacteria species (MOI = 100). Each scratch diameter was determined in five representative image sections for three technical replicates per experiment each 4 h up to 24 h from the beginning using the Biozero BZ 8000 (Keyence, Osaka, J) and corresponding Biozero software. Uninfected hDFSCs were used as control.

To visualize chlorophyllin uptake in E. coli, cells were cultured in LB medium in presence of 25 mg/L chlorophyllin for 20 min. Cells were harvested by centrifugation and washed with fresh LB medium twice before they were visualized using the Biozero BZ-8000 closed digital and inverted fluorescence microscope (Keyence, Osaka, Japan). Fluorescence images were taken with a 100× oil immersion objective, an excitation wavelength of 450–490 nm and an emission cut-off at 510 nm.

The appearance of the RCA product before and after EDTA treatment was observed under a fluorescent microscope (Biozero BZ-8000, KEYENCE, Osaka, Japan) after staining with SYBR-Gold (Molecular Probes, Eugene, OR, USA).

After differentiation, 3T3-L1 cells were washed twice with phosphate buffered saline (PBS, pH 7.4), fixed with lipid droplet assay fixative solution (Cayman kit) for 1 h, and then stained with Oil red O solution (in 60% isopropanol) at room temperature for 10 min. After washing, cells were checked and pictures were taken using a microscope (BioZero BZ-8000; Keyence, Osaka, Japan). Moreover, the dye was extracted with dye extraction solution and the absorbance (OD. 420 nm) was measured by a Spectra Max microplate reader (Spectra Max 190; Molecular Devices Corporation, CA, USA).