Phase contrast microscope

RAW 264.7 cells were treated with a series of I6CA concentrations or LPS (Sigma-Aldrich Chemical Co). After 24 h of incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay was performed as previously described (Hasnn et al. 2019 (link)). The LPS-treated cells were considered a positive control for activating the macrophages. The morphologic changes were observed under a phase-contrast microscope (Carl Zeiss, Oberkochen, Germany).

To see the effect of the introduced mutations on bacterial cell length, pictures were taken with a phase contrast microscope (Carl-Zeiss, Sliedrecht, The Netherlands) and analysed using ImageJ [27 (link)] with the Coli-Inspector plugin (Norbert Vischer, Bacterial Cell Biology, University of Amsterdam). This plugin is able to determine the cell length and diameter of individual cells in an automated manner. From this data a histogram was constructed and the median cell length was determined.

ADAM proteins were assayed in paraffin embedded cross-sections of rat testis fixed in Bouin’s solution (Sigma, St Louis, MO, USA). For antigen retrieval slices were treated with sodium citrate 0.01 M, pH 6.0 and heat until boil. The samples were treated with 3% H₂O₂ for 10 min, then, to prevent unspecific binding, a standard protein block System (Ultra V block, LabVision, Freemont, VA) was applied for 10 min. Primary antibody against ADAM17 (1 μg/ml) or ADAM10 (1 μg/ml), prepared in 3% BSA in TBS containing 0.1% Tween-20 (TBS-Tween), was applied to the slice and incubated overnight at 4°C in a humidified chamber after being washed three times for 10 min each in TBS-Tween. Biotinylated secondary antibody, streptavidin–biotinylated–peroxidase complex, amplification reagent (biotinyl tyramide) a peroxidase-conjugated streptavidin were applied step by step for 10 min each. Afterwards, slides were washed twice in a buffer for 3 min each. Finally, substrate-chromogen solution consisting of concentrated Tris–HCl and 0.8% H₂O₂ (substrate) and 3, 3-diaminobenzidine tetrahydrochloride (DAB) solutions (chromogen) were applied for 5 min and washed in distilled water. Samples were observed under a phase contrast microscope (Zeiss, Germany) and photographed with a digital camera (CoolPix 4500, Nikon, Japan).

The morphology and confluence of 4T1 cells were analyzed using a phase contrast microscope (Zeiss, Germany) and the AxioVision^® software (Zeiss, Germany).
Twenty-four hours after treatment (10 µg/mL), 4T1 cells were fixed using Karnovsky overnight. Samples were washed with cacodylate buffer (0,1 M), post-fixed with osmium tetroxide and dehydrated using graded acetone (30–100%). Then, they were critical-point-dried (Balzers, CPD 030, Germany) from liquid CO₂ and gold-sputtered. The cell morphology was observed using scanning electron microscopy.

The cell viability was examined using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetra-zolium bromide (MTT) assay according to the previous method [23 (link)]. In brief, 1 × 10⁴ cells per well were plated in 96-well plates. After 24 h, the cells were treated with the desired concentrations of genistein with or without N-Benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk, Calbiochem, San Diego, CA, USA), N-acetyl-L-cysteine (NAC, Invitrogen, Waltham, MA, USA) or LY294002 (Cell Signaling Technology, Inc., Danvers, MA, USA). After 48 h, the medium was changed with fresh medium containing 50 μg/mL MTT solution (Invitrogen). After 2 h, the medium was removed and added 100 μL of DMSO. Absorbance at 540 nm was measured using a microplate reader (Molecular Device Co., Sunnyvale, CA, USA). The morphological changes of cells following genistein treatment were observed and visualized by a phase-contrast microscope (Carl Zeiss, Oberkochen, Germany).