Cells of the immortalised mesenchymal stromal cell line N-KM (normal bone marrow) [41 ] were cultured in 6- or 24-well plates in 10% FBS, 100 U/mL penicillin, 100 U/mL streptomycin and 100 U/mL glutamine (Life Technologies) supplemented IMDM (Lonza, Cologne, Germany) until they reached approximately 70% confluency.
For the analyses of EV uptake equivalents of 1 × 108 particles of the EV-enriched samples were added to the cells. After incubation for 14–16 h at 37°C, the medium with residual particles was removed and fresh culture media were added. At first, cells were analysed by fluorescent microscopy on an Axio Observer.D1 microscope platform with Plan-Apochromat 20×/0.8 lenses (Zeiss, Oberkochen, Germany). To harvest cells for flow cytometric analysis, cells were treated with 0.25% trypsin (Lonza) for 5 min at 37°C. The enzymatic reaction was stopped by the addition of fresh culture media. Cells were pelleted by centrifugation for 5 min at 800 × g, re-suspended in isotonic solution for flow cytometry (Beckman Coulter) and analysed on a Cytomics FC500 flow cytometer (Beckman Coulter) for their eGFP-intensity. The mean fluorescence intensity was measured for all samples in comparison to untreated N-KM cells.
For the analyses of EV uptake equivalents of 1 × 108 particles of the EV-enriched samples were added to the cells. After incubation for 14–16 h at 37°C, the medium with residual particles was removed and fresh culture media were added. At first, cells were analysed by fluorescent microscopy on an Axio Observer.D1 microscope platform with Plan-Apochromat 20×/0.8 lenses (Zeiss, Oberkochen, Germany). To harvest cells for flow cytometric analysis, cells were treated with 0.25% trypsin (Lonza) for 5 min at 37°C. The enzymatic reaction was stopped by the addition of fresh culture media. Cells were pelleted by centrifugation for 5 min at 800 × g, re-suspended in isotonic solution for flow cytometry (Beckman Coulter) and analysed on a Cytomics FC500 flow cytometer (Beckman Coulter) for their eGFP-intensity. The mean fluorescence intensity was measured for all samples in comparison to untreated N-KM cells.
