Mitochondria were then diluted ten times with washing buffer and samples were centrifuged at 15000 g for 5 min at 4°C.
Cells from each group were washed one time with PBS and lysed in RIPA buffer; then the samples were centrifuged at 15 000 ×g for 10 min and the supernatants were collected and stored at −80°C.
The homogenates were then made isotonic by the addition of 0.4 mL of 1.46 M sucrose in buffer C. The samples were centrifuged at 10 000 g for 15 min at 4 °C.
After addition of triethylamine (TEA) buffer (pH 4.2), samples were centrifuged and supernatants were transferred to an amber vial for injection (60 μl) onto of a Zorbax-SB-C8 column (MacMod Analytical Inc., Chadds Fords, PA, USA) with a mobile phase of acetonitrile : 0.025 M TEA buffer (pH 4.2) (25 : 75%).
After more than 99% equilibrium between charge-buffer and ghosts, the samples were centrifuged (see above), charge-buffers were removed for scintillation counting (2 x 25 μl) and the ghosts were transferred to a new tube containing 5 ml buffer A without protein.
After 10 minutes, the red blood cells were lysed using 2 ml Pharm Lyse Buffer (BD Biosciences), and the samples were centrifuged for 5 minutes at 200×g at 20°C.
After the 10 min lysis period, phosphate buffered saline was added and samples were centrifuged, decanted, and vortexed before analysis by flow cytometry (C6, Accuri, Ann Arbor, MI, USA).
Briefly, hemispheres ipsilateral or contralateral to the intracranial TMEV injection site were homogenized in microvessel isolation buffer, 26% dextran was added and samples were centrifuged for 10 minutes at 6874 RPM.
Cells were collected and lysed in extraction buffer (Sigma-Aldrich, E1156), then the samples were centrifuged at 1000× g for 10 min and then at 2000× g for 20 min. The supernatant, containing the crude migrasome fraction, was fractionated at 150000× g for 4 h in a multi-step optiprep dilution gradient.
Samples were centrifuged and fixation buffer (0.5 ml) was added to the samples and incubated at room temperature for 20 min.
