Amicon ultra 2 10 kda centrifugal filter devices

Extracellular vesicles were isolated from 500 μL citrate plasma using qEV size‐exclusion chromatography Columns (IZON Science, Oxford, UK). The columns were equilibrated with 20 mL of 0.22‐μm‐filtered PBS/0.32% citrate (Greiner Bio‐One GmbH, Kremsmüster, Austria), and EVs were isolated according to the protocol of the vendor. Fractions of 500 μL were collected, and the eluted fraction numbers 7–10 were concentrated using Amicon Ultra‐2 10 kDa Centrifugal Filter Devices (Merck Millipore). All samples were stored at −80 °C. EV specimens from patients with invasive adenocarcinoma (i.e., rectal cancer) and noninvasive adenoma polyps were termed RC_EV and AP_EV, respectively.

Plasma EVs from individual samples were isolated using qEV size exclusion chromatography columns (IZON Science, Oxford, UK) according to the manufacturer’s protocol. Briefly, columns were equilibrated with 20 mL of 0.1 µm-filtered phosphate buffered saline (PBS) (Gibco, Life Technologies) before 400 µL of plasma was added and 500 µL fractions were collected. Fractions 7–9, containing the majority of EVs, were pooled and concentrated using Amicon Ultra-2 10 kDa Centrifugal Filter Devices (Merck Millipore). Similarly, for pooled plasma EV preparations of each patient group, a total of 1.4 mL plasma were pooled from 60 to 100 µL plasma per individual (HIV + T2D: n = 21, T2D: n = 14, HIV: n = 23, Controls: n = 23) and isolated in three runs on the qEV column. All samples were stored at − 80 °C.