Tecnai f20 twin transmission electron microscope

Chemicals or reagents were purchased from commercial sources and used without further processing unless otherwise stated. Research-grade foetal bovine serum (FBS, sterile triple 100 nm filtered) and DMEM cell-culture media were purchased from Fisher Scientific. MDA (MDA-MB-231 human breast adenocarcinoma) and MSTO (MSTO-211H human biphasic mesothelioma) cell lines were from ATCC (Manassas, VA). Sephadex G-25 Nap-5 columns were purchased from GE Healthcare. The ultra-centrifugation was performed on a Beckman Coulter Optima LE-80K ultra-centrifuge with an SW28 rotor. High-performance liquid chromatography (HPLC) was performed on an Agilent 1260 Infinity system equipped with an auto-sampler and a variable wavelength UV detector. The HPLC column was a size-exclusion column (Superose 12 10/300 GL from GE Healthcare) (9 μm-13 μm). The Gel Filtration HMW Calibration Kits were purchased from GE Healthcare and the liposome labelled with DiO (120 nm) for calibration was prepared in-house by Dr Alexander Klibanov. A NanoSight NS300 system (Malvern Instruments Inc., Westborough, MA) and a Tecnai F20 Twin transmission electron microscope (FEI, Hillsboro, OR) were used to characterize the vesicle samples.

Human Dicer-RNA complexes were assembled by pre-incubating 150 nmol/L hDicer with a 500% excess of unedited and edited pre-miR-151 in a specific volume containing 20 mmol/L Tris-HCl, pH 6.8, 25 mmol/L NaCl, 2 mmol/L MgCl_2, 1 mmol/L DTT and 2 mmol/L EDTA and 5% glycerol. All the samples were negatively stained on holey carbon grids covered by a thin layer of continuous carbon over holes with 2% (w/v) uranyl acetate solution. All micrographs were collected on a Tecnai F20 Twin transmission electron microscope (FEI) running at 200 kV or an Tecnai-12 Biotwin electron microscope (FEI) operated at 120 kV, using a nominal magnification of 50,000× or 49,000×, respectively. The micrographs were collected on Ultrascan 4000 CCD camera (Gatan Inc.) at specimen-level pixel sizes of 0.223 nm or 0.29 nm, respectively, using a dose of ~30 e⁻ Å⁻² and a nominal defocus range of −1 to −3 μm.

Cryo-TEM was performed by the molecular electron microscopy core at UVA. P20 and P100 fractions were resuspended in 30 mL PBS. An aliquot of the sample (~ 3.5 μL) was applied to a glow-discharged, perforated carbon-coated grid (2/1-3C C-Flat; Protochips, Raleigh, NC), manually blotted with filter paper, and rapidly plunged into liquid ethane. The grids were stored in liquid nitrogen, then transferred to a Gatan 626 cryo-specimen holder (Gatan, Warrrendale, PA) and maintained at ~ 180 °C. Low-dose images were collected on a Tecnai F20 Twin transmission electron microscope (FEI {now ThermoFisher Scientific}, Hillsboro, OR) operating at 120 kV. The digital micrographs were recorded on a TVIPS XF416 camera (Teitz, Germany).