Macs separator

Strains were grown to OD₆₀₀ 0.8 in synthetic dropout media lacking appropriate auxotrophic selection markers. A total of 200 ml of each strain was harvested, and the cell pellet was stored at −80°C. Pellets were thawed and resuspended in 3 ml lysis buffer (25 mM Tris-HCL pH 7.5, 50 mM KCl, 10 mM MgCl₂, 2 mM DTT, 5% glycerol, 1 tablet/20 ml cOmplete EDTA-free protease inhibitor cocktail [Roche]), then crushed in a mortar with pestle under liquid nitrogen. Thawed lysates were centrifuged at 500 g for 5 min at 4°C and the supernatant was transferred to a new tube and centrifuged at 500 g for 5 min at 4°C. Protein concentration of the lysate was determined with Pierce BCA protein assay (Thermo Fisher Scientific) and 3 mg of total protein was incubated with 50 µl µMACS Anti-GFP MicroBeads (Miltenyi Biotec) while shaking for 1 h at 4°C. Samples were loaded onto µ Columns (Miltenyi Biotec) in a µMACS Separator (Miltenyi Biotec) and washed twice with 300 µl TEV cleavage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM DTT). About 25 µl TEV cleavage buffer containing 5 µg TEV protease (custom-made) was added onto the columns and incubated for 1 h at room temperature. Input and eluate samples were mixed with NuPAGE LDS Sample Buffer (Thermo Fisher Scientific), incubated at 70°C for 5 min, and stored at −20°C until analysis by Western blotting or mass spectrometry.

Cells were cultured in T175 flasks and at 80% confluency cells were washed once with cold DPBS and then incubated with 2 mM of dithiobis(succinimidyl propionate (Pierce™ Premium Grade DSP, Thermo Fisher Scientific) for 1 h at 4 °C. Then, quenching buffer (1 M Tris, pH 7.5) was added for 15 min at 4 °C. Cells were lysed in 1.2 mL of Pierce ™ IP Lysis Buffer (Thermo Fisher Scientific) containing 1% (v/v) Halt™ protease and phosphatase inhibitor cocktail (Thermo Fisher) followed by one wash with cold DPBS. Cell lysates were centrifuged and stored as described above. For the immunoprecipitation 500 µg of protein was incubated with 2–4 µg of primary or nonspecific IgG isotype control antibodies (Supplementary Table S3) overnight at 4 °C on HulaMixer™ Sample Mixer with rotating angle at 45° and speed at 25 rpm (Thermo Fisher). Lysate-antibody complex was then incubated with 100 µL of μMACS Protein G MicroBeads (Miltenyi Biotec) for 1 h at 4 °C using HulaMixer™ Sample Mixer at 25 rpm. Postabsorptive supernatant was magnetically isolated after eluting the lysate-antibody-bead complex through µMACS™ separator and µ column (Miltenyi Biotec) according to the manufacturer's instructions. Epitope location and specificity of the in-house antibodies against sortilin and syndecan-1 used in this method are provided in Supplementary Figs. S12 and S13.

The 0.5 mL samples of serum and plasma were diluted with an equal volume of HBSS and centrifuged at 10,000× g for 45 min. Subsequently, EVs were captured using the Exosome Isolation Kit Pan (MicroBeads conjugated to CD9, CD63, and CD81) with µColumns and µMACS separator, according to the manufacturer’s protocol (all Miltenyi Biotec, Bergisch Gladbach, Germany). EVs were eluted in 120 µL HBSS + PI.

GST pull-down assay was performed incubating 500 ng His-HSP27 (Calbiochem) with 500 ng of GST-NLK recombinant protein (Abnova) in 400 µl binding buffer (20 mM HEPES, pH 7.5, 0.01% NP-40, 0,01% BSA, 100 mM KCl, 5 mM DTT) with agitation for 3 hour at 4°C. Thereafter 30 µl µMACS anti-GST MicroBeads (Miltenyi Biotec) was added and following 30 minutes incubation at 4°C, protein separation was performed on a μ Column in a magnetic field of µMACS Separator (Miltenyi Biotec) according to manufacturer's protocol. GST pull-downs were analyzed with SDS-PAGE and Western blotting.

PgDCEXO were incubated with microbeads that recognize CD81, CD9 and CD63 positive exosomes (pan EXO) (Miltenyi Biotech Auburn, CA, USA). Magnetic separation of labeled EXO was performed by loading it onto a µ column placed in a magnetic field of µMACS separator (Miltenyi Biotech Auburn, CA, USA). The magnetically labeled EXO are retained within the column and the unlabeled vesicles run through the column. The positively selected retained EXO are then eluted from the column. The number of particles in the run through (passage1) of unlabeled extracellular vesicles (EV) fraction was then counted using NTA. Magnetic separation of the run through was repeated (passage 2) to completely deplete pan EXO from the run through and then particles counted in the final run through of unlabeled EV and a percentage to the total number of EV was calculated. The eluate (CD81, CD9 and CD63 positive exosomes) and the run through were then analyzed using WB.

The lung endothelial cells were isolated from whole lung tissues using CD45 (Miltenyi Biotec, 130-052-301) and CD31 Microbeads (Miltenyi Biotec, 130-097-418) as previously described [22 (link)]. In brief, the harvested lungs were minced and digested with collagenase II (Gibco, 17101015) and filtered through a 100-μm nylon mesh. The cell suspension was resuspended in bovine serum albumin and incubated with anti-CD45-conjugated magnetic beads for negative selection, followed by positive selection with anti-CD31-conjugated magnetic beads. The CD31-positive cells were collected with an MACS separator (Miltenyi Biotec) following the manufacturer’s protocols. The purity of the endothelial cell population was confirmed by immunofluorescence of anti-CD31 (Abcam, ab7388) and anti-VE-cadherin (Affinity, AF6265). The primary mouse lung endothelial cells (MLECs) from 8-week-old male mice were cultured for in vitro experiments. Isolated MLECs were cultured with Dulbecco’s modified Eagle’s medium (DMEM) containing endothelial growth supplement, and the cells between passages 2 and 6 were used for in vitro experiments. The freshly isolated lung endothelial cells from lean and DIO mice were collected immediately to detect the protein levels by western blot.