cell type: HUVEC (human umbilical vein endothelial cells) treatment: after 24hrs treatment with PTC
Treatment protocol
After cells reached confluency, cells were washed several times with serum-free DMEM and then incubated in serum-free media for 24h to accumulate sEVs at 37°C, 5% CO2. Culture mediums were collected and centrifuged for 15min at 1000 rpm at 4°C. Subsequently, the culture supernatants were centrifuged for 45min at 4000rpm (4°C) and then passed through 0.2µm syringe filters (Filtropur S, #83.1826.001, Sarstedt). The supernatant was concentrated by ultrafiltration at 2000 rpm and 4°C using Vivaspin Turbo 15 (100,000 MWCO) units (Sartorius, #VS15T41) until a volume of 1ml was reached. The concentrated supernatant was transferred into Lo-Bind 1.5ml tubes and 0.5 volumes (V) of supplied buffer A was added. After incubation overnight at 4°C the samples were centrifuged for 1hr at 14000 rpm and 4°C. The supernatant was discarded, and the pellet was resolved in 100µl sterile (0.2µm) filtered PBS. For size exclusion chromatography Exo-spin columns were centrifuged for 30 seconds at 500 rpm after cap and stopper were removed. The columns were equilibrated by centrifugation at 500 rpm for 30sec using 200µl sterile filtered PBS. The sEV pellet solved in PBS was pipetted onto the column and centrifuged for 1min at 500rpm. The eluate was discarded, and the column was placed in a fresh 1.5ml-Lo-Bind tube. For elution, 200µl sterile filtered PBS was added to the column and centrifuged for 1min at 500 rpm. The sEV eluate was stored at -80°C. The surface protein concentration was determined using Bradford Protein Assay (Bio-Rad, #5000006).
Growth protocol
Human umbilical vein endothelial cells (HUVEC, ATCC, CRL-1730) were maintained at 37°C and 5% CO2 in endothelial cell growth medium MV2 (PromoCell, #C-22022)
Extracted molecule
total RNA
Extraction protocol
For purification of total RNA including miRNA 700µl QIAzol was added to sEVs derived from cell culture supernatants or serum/plasma. Subsequently, isolation was performed using the miRNeasy mMicro Kkit (Qiagen, #217084)
Label
Cy3
Label protocol
Samples were processed with the miRNA Complete Labeling & Hybridization Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's guidelines
Hybridization protocol
Agilent protocol
Scan protocol
Slides were scanned using a G2565CA microarray scanner (Agilent Technologies, Santa Clara, CA, USA).
Data processing
Pre-processing of expression data was performed according to Agilent's standard workflow. Using 5 quality flags (gIsPosAndSignif, gIsFeatNonUnifOL, gIsWellAboveBG, gIsSaturated, and gIsFeatPopnOL) from the Feature Extraction software output, probes were labelled as detected, not detected, or compromised. Gene expression levels were background corrected, and signals for duplicated probes were summarized by the geometric mean of non-compromised probes. After log2 transformation, a percentile shift normalization at the 75% level was performed.
