Macs magnetic μ column

The following antibodies were employed for immune-isolation of EVs: mouse anti-CD9 (Millipore, CBL162), mouse anti-CD63 (Abcam, ab8219), mouse anti-CD81 (BD, 555675). Briefly, EVs purified from approximately 4.5 × 10⁸ cells by differential centrifugation were resuspended in 100 μL of PBS, split equally between 4 eppendorf tubes (25 μL each), and resuspended in 500 μL PBS. Each sample was mixed with 2 μg of normal mouse IgG and 50 μL of MACs microbeads (Miltenyi Biotec) for 1 h and then pre-cleared through a MACs magnetic μ Column (Miltenyi Biotec). Flow through from each of the samples was collected, mixed with 2 μg of antibody against 1 of 3 different EV-associated tetraspanins or normal mouse IgG, 50 μL of MACs microbeads (Miltenyi Biotec) and left to capture overnight at 4^oC. The following day, bound EVs were captured separately on MACs magnetic μ Columns, washed with 1500 μL PBS, and then eluted with 100 μL of hot 2x sample buffer. Samples were then resolved via SDS-PAGE and immunoblotted for LC3 and EV marker proteins.