Axiovert 100

Cell migration was evaluated by the wound healing assay. Confluent cultures in 6-well plates of NFATc2 shRNA transfectants and control transfectants, or of melanoma cell line 71 were wounded using a sterile 200 μl pipette tip, then washed three times. AM404, 10058-F4, Siomycin A, or GSK126 inhibitors were then added and wound closure was assessed at 48 h by imaging wounds at ×5 through an Axiovert 100 microscope (Zeiss, Oberkochen, Germany) equipped with a digital camera (AxioCam MrC5, Zeiss). Wound width was evaluated through the TScratch software [49 (link)]. Data were expressed as percentage of closure of the original (t = 0 h) wound width. The invasive activity of melanoma cells was tested using the CultrexCoat BME Cell Invasion Assay 96 well (R&D Systems, MN, MN, USA), according to the manufacturer’s instructions. In these experiments melanoma cells were pre-treated for 48 h with different inhibitors as described for the migration assay. Fluorescence signal from calcein-labeled cells was read by an Infinite 1000 instrument (Tecan). Data were expressed as % of invasion by referring to a standard curve.

After IVF, all oocytes were gently washed in in vitro culture (IVC) medium (Ham's F-10 supplemented with 5% FBS, 0.11 mg/mL sodium pyruvate, 0.075 mg/mL L-glutamine, 0.6 mg/mL gentamicin) to remove spermatozoa and residual cumulus cells with the help of a stripper micropipette (The Stripper, BioTipp, Waterford, Ireland) equipped with 155 and 125 μm tips (RI-Tip, Gynemed, Lensahn, Germany). Presumptive embryos were moved to 20 μL microdrops of IVC medium covered by mineral oil in Petri dishes, where they were cultured singly for up to 8 days in a controlled atmosphere (39°C, 5% CO₂, and 5% O₂). The medium was not changed during embryo culture. During IVC, assessment of embryo development was performed every 24 h under an inverted microscope at 200 × magnification (Axiovert 100, Zeiss).

The cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescence was performed as recommended in the antibody data sheet. An epifluorescence microscope (Axiovert100, Carl Zeiss, Berlin, Germany) connected to a digital camera (SPOT RT Slider, Diagnostic Instruments, Sterling Heights, MI, USA) with SPOT software (Universal Imaging Corp., Marlow Buckinghamshire, UK) was used for image acquisition. Where indicated, image analysis was performed using the ImageJ software with the NeuronJ plugin.

In order to investigate the cell migration inhibition efficiency of the extracts, the wound healing assay was performed in MDA-MB-231 cells as described in [37 (link)] with some modifications. Briefly, cells (3x10⁵ cells per well) were seeded in a 6-well plate and allowed to grow overnight at 37°C and 5% CO₂ to form a confluent monolayer. After 24 h of serum starvation, linear ‘scratch’ wounds were created on the monolayer using a sterile cell scrapper. The cells were washed gently with PBS twice to remove cellular debris and detached cells. Subsequently, the cells were treated with the extracts (31.25 to 1000 μg/mL). The cells in negative control wells were incubated with complete culture medium. The plates were incubated at 37°C and the wound was photographed at 0, 12, and 24 h. At each time point, the distance over which the cells migrated was measured. Photographs were captured using an inverted phase contrast microscope (Zeiss Axiovert 100) equipped with a digital camera and the data were analyzed using NIH Image J software. Wound closure rates were then calculated quantitatively as the difference between wound width at 0, 6, and 12 or 24 h. The results were expressed as percentage cell migration. Three independent experiments were performed for each sample.

Morphology of MDA-MB-231 cells treated with or without S. ferruginea extracts was compared to determine the changes induced by the extracts. MDA-MB-231 cells were plated in 12-well plates and exposed to increasing concentrations (31.25 to 1000 μg/mL) of S. ferruginea extracts for 24, 48, and 72 h. The morphological changes were observed using inverted phase contrast microscope (Zeiss Axiovert 100) at 20 X magnification.

HVEC cells were cultured on 35 mm glass coverslips until 60–80% of confluency and incubated at 37 °C with exosome preparations or starvation medium as described above. Cells were then treated as previously described (Di Noto et al.24 (link), nuclear staining). Primary antibody: anti-NfkB, (Santa-Cruz). Secondary antibodies: Alexa Fluor 488 and DAPI. Fluorescent microscopy was performed on a ZEISS Axiovert 100 fluorescent microscope using a 63x oil immersion objective. NfkB nuclear and cytoplasmic fluorescence intensity quantification was determined using Image J on at least 100 cells for each treatment. Background was subtracted to nucleus and cytosol signal and ratio between Nuclear/Cytosolic fluorescent signal was calculated.