Bf3 meoh 14

We used the modified method of Moilanen [31 (link)], that is itself a modification of the method described by Folch [32 (link),33 (link)]. Fatty acids extraction and preparation of fatty acid methyl esters(FAME) from red blood cells (RBC) and tissue samples were carried out as previously described [16 (link),34 (link)]. Briefly, total lipids from phospholipids of RBC membranes were extracted by adding 0.9 mL of an acidified salt solution (H₂SO₄ 2 × 10⁻⁴ M, NaCl 0.1%). For fatty acids extraction from tissues, about 20 mg wet tissues were homogenized with 0.8 mL of ice cold 0.9% NaCl. All samples received 5.0 mL of chloroform:methanol (2:1, v/v) (Sigma-Aldrich, Milan, Italy) and the samples were mixed thoroughly and centrifuged at 1000× g for 10 min. The lower layer, containing fatty acids, was removed with care, replaced in a new tube and dried by a centrifugal evaporator (Bio-Rad, Milan, Italy). The FAME were obtained by adding toluene and BF₃·MeOH 14% (Sigma-Aldrich, Milan, Italy) and incubating for 2 h at 80 °C. After the addition of toluene and 5% aqueous sodium chloride solution, the samples were centrifuged at 470× g for 10 min. Fatty acid methyl esters contained in the upper layer of the tubes, were collected and transferred into a vial and analyzed.

We used the modified method of Moilanen [17 (link)], that is itself a modification of the method described by Folch [18 ]. Each sample of red blood cells (RBC) was thawed bringing to room temperature. Fatty acids were hydrolyzed from phospholipids of RBC membranes by adding 0.9 ml of an acidified salt solution (H₂SO₄ 2·10⁻⁴ M, NaCl 0.1%). Afterwards, were added 5.0 ml of chloroform: methanol (2:1, v/v) (Sigma-Aldrich, Milan, Italy) and the samples were mixed thoroughly and centrifuged at 1000 × g for 10 min. The lower layer, containing fatty acids, was removed with care, replaced in a new tube and dried by a centrifugal evaporator (Bio-Rad, Milan, Italy). Preparation of fatty acid methyl esters (FAME) was carried out by adding 1 ml of toluene and 1.5 ml of BF_3·MeOH 14% (Sigma-Aldrich, Milan, Italy) and incubating for 2 h at 80 °C. To the samples were added 2.5 ml of 5% aqueous sodium chloride solution, 1.5 ml of toluene and then centrifuged at 470×g for 10 min. The upper layer, containing FAME, was collected and transferred into a vial and analyzed.

The FA profile in membranes of exosomes and hybrid exosomes of all subjects was evaluated using the Moilanen method [25 (link)], a modified form of the method used by Folch [26 ]. Each sample of exosomes was thawed to room temperature. FAs were hydrolyzed from phospholipids of exosome membranes by adding 450 µL of an acidified salt solution (H₂SO₄ 2 × 10⁻⁴ M, NaCl 0.1%). Then, 2.25 mL of a chloroform: methanol (2:1, v/v) mixture were added (Sigma-Aldrich, Milan, Italy) and the samples were mixed and centrifuged at 1000 × g for 20 min. The lower layer, containing FAs, was gently removed, placed in a clean tube and dried with a centrifugal evaporator (Bio-Rad, Milan, Italy). Then, FA methyl esters (FAME) were prepared by adding 250 µL of toluene (Sigma-Aldrich, Milan, Italy cod.24511) and 750 µL of BF₃•MeOH 14% (Sigma-Aldrich, Milan, Italy cod 9005-64-5) and incubating for 2 h at 80 °C. In each sample, 1.250 mL of 5% aqueous sodium chloride solution and 250 µL of toluene were added and the resulting mixtures were centrifuged at 470 × g for 10 min. The FAME, being the upper layer, was collected, transferred into vials and analyzed.