4.11. Extracellular vesicle isolation and purification

All relevant data have been submitted to the EV‐TRACK knowledgebase (EV‐TRACK ID: {"type":"entrez-nucleotide","attrs":{"text":"EV190053","term_id":"151279693","term_text":"EV190053"}}EV190053) (Van Deun et al., 2017). Extracellular vesicles were isolated from supernatants collected from mock or Mtb‐infected THP‐1‐derived macrophages after 72 h of culture. Supernatants were centrifuged at 400 × g for 5 min and at 2000 × g for 10 min to exclude cells and cell debris, respectively. Debris‐cleared conditioned medium was then concentrated by 100 kDa ultrafiltration using regenerated cellulose Amicon Ultra (Millipore) at 2000 × g for 35 min, obtaining typically 250 μl concentrated conditioned medium. For tracking purposes, extracellular vesicles were stained with 5,6‐Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE; Invitrogen) by incubating the concentrated conditioned medium with 100 μM CFSE for 2 h at 37°C (Morales‐Kastresana et al., 2017). CFSE positive or unstained extracellular vesicles were then isolated from the concentrated conditioned medium (and washed away from free CFSE dye) by size‐exclusion chromatography (SEC) using a modification of a previous method (Monguió‐Tortajada et al., 2017). Briefly, 12 ml of Sepharose CL‐2B (Sigma‐Aldrich) were extensively washed with PBS (Oxoid) and packed in a Puriflash dry load empty 12G flash column (Interchim‐Cromlab). Concentrated conditioned medium was loaded into the SEC, and 500 μl fractions (up to 35) were collected immediately after eluting with PBS. Protein elution was checked by reading absorbance at 280 nm of each fraction using Nanodrop (Thermo Scientific). The presence of extracellular vesicles in the SEC fractions was determined according to their content in tetraspanins by bead‐based flow cytometry, as previously described (Monguió‐Tortajada et al., 2019). Briefly, extracellular vesicles were coupled to 4‐μm aldehyde/sulphate‐latex microspheres (Invitrogen) for 15 min, blocked overnight with BCB buffer (PBS/0.1% BSA/0.01% NaN₃; all from Sigma‐Aldrich) and spun down at 2000 × g for 10 min. Extracellular vesicles‐coupled beads were then labelled with the primary antibodies anti‐CD9 (Clone VJ1/20) and anti‐CD63 (Clone TEA3/18) at 1:100 dilution (kindly provided by Dr. María Yáñez‐Mó from UAM; CBM‐SO, IIS‐IP and Dr. Francisco Sánchez‐Madrid from Hospital Universitario de la Princesa, IIS‐IP, UAM, CNIC) and secondary antibodies Cy5‐conjugated Donkey anti‐Mouse or A647‐conjugated Goat F(ab')2 Anti‐Mouse IgG (Jackson ImmunoResearch), performed at RT for 30 min under mild shaking, washed after each step with BCB buffer and centrifuged at 2000 × g for 10 min. Data were acquired in a FACSLyric flow cytometer (BD) and analyzed by FlowJo v.10.2 software (BD). Extracellular vesicles‐containing fractions were pooled together and adjusted to the desired volume with PBS using 100 kDa‐ultrafiltration 2 ml‐Amicon Ultra (Millipore). Extracellular vesicles were kept at 4°C and used within 24 h for the in vitro experiments or frozen (‐1°C/min) at ‐80°C.