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EV and whole-cell lysis

JB Julia Bauzá-Martinez
AH Albert J. R. Heck
WW Wei Wu
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JY EVs and WC were lysed on an end-to-end rotating platform at 4 °C in Pierce IP Lysis Buffer (ThermoFisher) supplemented with 1× complete protease inhibitors (Sigma-Aldrich,USA), 50 μg/mL DNase Ι (Sigma-Aldrich), and 50 μg/mL RNase A (Sigma-Aldrich). EVs were lysed in 200 μL of lysis buffer for 30 min followed by 20 cycles of sonication at 4 °C (30 s on, 30 s off) in a Bioruptor (Diagenode, Belgium). Cells were lysed in 10 mL of lysis buffer for 1 h. The lysates were spun down at 20,000 g at 4 °C for 1 h and the aqueous phase containing the solubilized proteins was kept. Lysate protein concentrations were determined with a microplate BCA-protein assay (ThermoFisher). Aliquots of 15 μg were taken for further characterization and all the samples were snap-frozen until further processing.

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