Morada ccd camera

FEMX-I cells were grown for 1–2 days in 35-mm Petri dishes. Cells were then pre-fixed by addition of an equal volume of 4% paraformaldehyde (PFA) into the cell culture medium. After one hour, cells were fixed in 4% PFA, 0.1 M CaCl₂ with or without 1% glutaraldehyde in phosphate buffer overnight at room temperature or at 4 °C. After washing, cells were incubated in 1% aqueous osmium tetroxide for one hour at room temperature. Cells were then washed with water and contrasted with 0.5% aqueous uranyl acetate for 30 min at room temperature. Afterward, samples were processed through a graded series of ethanol for standard plastic embedding in LX 112 resin (LADD Research, VT). Sheets of resin embedded cells were removed from the Petri dishes by liquid nitrogen. Sections were cut at 70-nm on an UCT ultramicrotome (Leica Microsystems) parallel to the cell support (Petri dish), and post-stained with uranyl acetate and lead citrate. The samples were viewed in a Morgagni or Tecnai Biotwin T12 electron microscope (Thermo Fisher Scientific, former FEI, former Philips) and images acquired on a Morada CCD camera (EMSIS Münster Germany, former Olympus, former SIS) or F416 CMOS camera (TVIPS, Gilching, Germany).

EVs were isolated from individual plasma as described above. For TEM, the EV pellets were fixed with 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.0) for 1 h at 4 °C. The EVs were then resuspended in 100 mM sodium cacodylate buffer (pH 7.0), placed on to a grid with a glow discharged carbon support film and stained with 2% aqueous Uranyl Acetate (Sigma-Aldrich, Gillingham, UK). Individual EVs were imaged by TEM using a Morada CCD camera (EMSIS GmbH, Münster, Germany) and processed via iTEM (EMSIS).

A suspension of isolated OMVs/MVs (1.4 × 10⁸ vesicles/ml) was used for TEM imaging. Mesh copper grids were prepared with glow discharged carbon support films and 10 μl of OMV/MV samples applied to the grid and incubated for 2 min. The grids were then washed five times with 50 μl of 1% aqueous uranyl acetate. The last drop was left to incubate on the grid for 1.5 min before being wicked off by torn filter paper. Grids were left to dry for 5 min before being viewed. Micrographs were taken with a JEOL JEM 1230 transmission electron microscope (JEOL, Japan) operated at 80 kV at a range of magnification mainly around a magnification of 80,000–100,000. Digital images were recorded on a Morada CCD camera (EMSIS, Germany) and processed via iTEM (EMSIS, Germany).

The EV pellets obtained from plasma, as described above for each individual, were fixed with 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.0) for 1 h at 4 °C. EVs were then resuspended in 100 mM sodium cacodylate buffer (pH 7.0) and placed on to a grid with a glow-discharged carbon support film. The EVs were stained with 2% aqueous Uranyl Acetate (Sigma-Aldrich, Gillingham, UK) and imaged by using transmission electron microscopy (TEM) with a Morada CCD camera (EMSIS GmbH, Münster, Germany), processed via iTEM (EMSIS).

A suspension of isolated MVs (1.4 × 10⁸ MVs/ml) was used for TEM imaging. MV samples (10 μL) were applied to mesh copper grids, prepared with glow discharged carbon support films, and incubated for 2 min. The grids were then washed five times with 50 μl of 1 % aqueous uranyl acetate. Grids were left to dry for 5 min before being viewed. Micrographs were taken with a JEOL JEM 1230 transmission electron microscope (JEOL, Japan) operated at 80 kV at a magnification of 80,000 to 100,000. Digital images were recorded using a Morada CCD camera (EMSIS, Germany) and processed via iTEM (EMSIS).