Journal: PLoS ONE
Article Title: Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles
Figure Lengend Snippet: Detection of Venus-fluorescence and A . t .-sequences in recipient cells after passaging. (a) 2x10 5 hMSC were seeded into T25, incubated overnight to reach adherence (d0) and fed with EV derived from A . t .-hMSC cultures for 2 weeks. (b) Venus-positive cells were detected in 2 of 4 flasks (d14). One flask with 7 positive cells was passaged into 4xT25 flasks. (c) 7 days later (d21), one flask contained 10 Venus-positive cells. This culture was expanded again into 4xT25. (d) Venus-positive cells at d28 were evident in 2 flasks out of 4 with 13 cells in one flask and 1 cell in the second flask. Exemplarily, one positive MSC spot with corresponding phase-contrast for each time point is shown. Magnification x 100. (e, f) DNA of the flask with 13 Venus-positive cells was pretested in nested SYBR Green-based qPCR. Out of 10 primary reaction tubes, 4 were positive in the nested qPCR tested in 8 replicates (tubes 2, 4, 7 and 8; not shown) and were retested in TaqMan-based qPCR (e) and ddPCR (f). Shown are the results for positive control (pc, 10 copies/PCR reaction; 4 replicates in TaqMan-based qPCR and 2 replicates in ddPCR), negative control (nc, untransduced hMSC; 8 replicates in TaqMan-based qPCR and 2 replicates in ddPCR), and tube 2 and 4 (16 replicates in TaqMan-based qPCR and ddPCR).
Article Snippet: EV-transfer to recipient cells Recipient human BM-hMSC (2.5-5x104 ) unrelated to hMSC used for A .t .-transduction were seeded into T25-flasks (Greiner).
Techniques: Fluorescence, Passaging, Incubation, Derivative Assay, SYBR Green Assay, Real-time Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction, Negative Control