90ti fixed angle rotor

The cells were cultured in BPE- and exosome-free KSFM for 72 h upon 80% confluency. The conditioned medium was collected and centrifuged at 500× g (Centrifuge 5417, Eppendorf, Hamburg, Germany) for 5 min at 4 °C to remove any dead cells. The supernatant was collected and re-centrifuged at 2000× g for 10 min at 4 °C to remove any remnants from the cell. The following supernatant was then filtered using a 0.22 µm filter (Merck Millipore, Burlington, MA, USA), ensuring only small molecules passed through the filter. Conditioned medium was collected (approximately 32 mL) in OptiSeal™ tubes (Beckman Coulter, Brea, CA, USA) and ultracentrifuged at 100,000× g (90Ti fixed angle rotor, Beckman Coulter, Brea, CA, USA) for 2 h at 4 °C. The pellet obtained following ultracentrifugation was re-suspended in sterile phosphate-buffered saline (PBS, Gibco™ ThermoFisher Scientific, Waltham, MA, USA) to wash out any potential media remains. The suspension was re-centrifuged with the same specifications and the resulting pellet was re-suspended in 200 µL of PBS. The obtained EV sample in PBS was stored at −80 °C for further experiments.

Blood was sampled from 12 healthy volunteers after obtaining written informed consent. The study protocol and all study procedures were reviewed and approved by the local ethics committee (Number 21-523 ex 09/10). All participants gave written informed consent before being enrolled into the study. Serum from four healthy controls was pooled for HDL isolation and storage experiments were repeated three times. Serum density was adjusted with potassium bromide (Sigma, Vienna, Austria) to 1.24 g/ml and a two-step density gradient was generated in centrifuge tubes (16 × 76 mm; Beckman) by layering the density-adjusted plasma (1.24 g/ml) underneath a NaCl-density solution (1.006 g/ml), as described (26 (link)). Tubes were sealed and centrifuged at 415,000 g for 6 h in a 90Ti fixed angle rotor (Beckman Instruments, Krefeld, Germany). After centrifugation, the HDL-containing band was collected, desalted via PD10 columns (GE Healthcare, Vienna, Austria), and immediately used for experiments.

The protocol for EV extraction was described earlier by Parekh et al. [21 (link)] and Ramos et al. [35 (link)]. Briefly, upon confluence, HCEC-12 cells were serum starved for 72 h. The medium was centrifuged at 500 rpm for 5 min at 4 °C (centrifuge 5417, Eppendorf, Hamburg, Germany) followed by 2000 rpm for 10 min at 4 °C, sequentially. The resulting supernatant was filtered through a 0.22 micrometer filter (Merck Millipore, Burlington, MA, USA) and transferred to OptiSeal tube (Beckman Coulter, Brea, CA, USA). The supernatant was ultracentrifuged at 49,000 rpm (100,000× g) using a 90Ti fixed angle rotor (Beckman Coulter, Brea, CA, USA) for 2 h at 4 °C. The pellet was re-suspended in PBS, followed by a second ultracentrifugation. The final pellet was re-suspended in the media, PBS or Trizol at −80 °C for further experiments. For the measurement of small RNA concentration (Agilent Bioanalyzer Small Assay using Bioanalyzer 2100 Expert instrument—Agilent Technologies, Santa Clara, CA, USA), 1 μL of total EV-RNA was utilized. NGS libraries were generated as described earlier [21 (link),35 (link)], and the sequencing was performed on Illumina NextSeq 500 platform. DEseq was used for abundance determination and differential expression. TarBase v7.0 of KEGG (mirPath v.2, Diana tools) was used for pathway analysis.

Serum density was adjusted with potassium bromide (Sigma, Vienna, Austria) to 1.24 g/ml, and a two-step density gradient was generated in centrifuge tubes (16 × 76 mm, Beckman, Nr. 342,413) by layering 3 ml density-adjusted plasma (1.24 g/ml) underneath a KBr-density solution (1.063 g/ml) as described (14 ). Tubes were sealed and centrifuged at 65.000 rpm (415.000 g) for 6 h in a 90Ti fixed angle rotor (Beckman Instruments, Krefeld, Germany). After centrifugation, the HDL-containing band was collected, desalted via PD10 columns (GE Healthcare, Vienna, Austria) and either immediately used for experiments or stored with 5% sucrose at −70°C (15 (link)).

HDL was isolated from serum either by a two-step density gradient ultracentrifugation method17 (link) or via dextran sulfate precipitation. For the two-step density gradient ultracentrifugation method, serum density was adjusted with potassium bromide (Sigma, Vienna, Austria) to 1.24 g/ml and a two-step density gradient was generated in centrifuge tubes (16 × 76 mm, Beckman) by layering the density-adjusted plasma (1.24 g/ml) underneath a NaCl-density solution (1.006 g/ml) as described17 (link). Tubes were sealed and centrifuged at 65.000 rpm (415.000 g) for 6 hours in a 90Ti fixed angle rotor (Beckman Instruments, Krefeld, Germany). After centrifugation, the HDL-containing band was collected, desalted via PD10 columns (GE Healthcare, Vienna, Austria) and immediately used for experiments.
For HDL isolation from serum by dextrane sulfate, we used a commercial available kit from Cell Biolabs (Nr.: STA-607). The isolation was performed according to the manufacturer’s instructions.