Multi a holey carbon film

Samples were prepared by pipetting 5 μL of liposomes on a Quantifoil Multi A holey carbon film supported by a copper grid (Quantifoil Micro Tools GmbH, Jena, Germany). The samples were immediately frozen by immersion into liquid ethane, then transferred under liquid nitrogen to a FEI Tecnai G2 F20 microscope (FEI Company, Hillsboro, OR). Images were captured at approximately − 170 °C with a Gatan CCD camera (Gatan Inc., Warrendale, PA) and a 200 kV acceleration voltage.

EVs isolated from 4 × 10⁴ infected monocytes (MOI 1:1) were collected in 4-µL suspension, and were applied onto copper EM-grids covered by a QUANTIFOIL Multi A holey carbon film (Quantifoil Micro Tools), and excess of liquid was blotted automatically between two strips of filter paper. Subsequently, the samples were rapidly plunge-frozen in liquid ethane (cooled by liquid nitrogen at about −180 °C) in a cryobox (Carl Zeiss). Excess of ethane was removed with a piece of filter paper. The samples were transferred immediately using a Gatan 626 cryo-transfer holder (Gatan) into a pre-cooled cryo-electron microscope (Philips) operated at 120 kV and imaged under low dose conditions. The images were recorded with a 2k F216-CMOS-camera (CMOS-camera and acquisition software, EMMANU4 v 4.00.9.17, TVIPS). In order to minimize the noise, four images were recorded and averaged to one image.