Syto rnaselect green fluorescent cell stain

At 48 hours posttransfection incubation, cells were loaded into temperature- and CO2-controlled live-cell imaging chamber of Leica SP8 confocal microscope. Cells were imaged typically by use of 2 laser wavelengths (488 nm for Cry2 activation and 560 nm for mCherry imaging). Time series images for demixing index plots were taken every 2 seconds for at least 300 seconds with 488 nm illumination. FRAP assay was carried out by use of FRAP wizard in LAS-X software. Designated regions (region of interests, (ROIs)) were photobleached with UV for 60 seconds, followed by time series imaging to observe fluorescence recovery. Fluorescent intensities from ROIs were measured with ImageJ software (NIH). For live-cell DNA and RNA staining, Hoechst 33342, Trihydrochloride, Trihydrate (ThermoFisher Scientific, H3570), and SYTO RNASelect Green Fluorescent Cell Stain (ThermoFisher Scientific, S32703) was treated for 20 minutes according to manufacturer’s protocol and washed 3 times with complete media, followed by confocal imaging.

NoLimits DNA fragments, YOYO-1 Iodide, and SYTO RNASelect Green Fluorescent Cell Stain were purchased from Thermo Fisher Scientific (Waltham, MA, United States). Human genomic DNA (gDNA) was purchased from Promega Corporation (Madison, WI, United States). Endotoxin-free Dulbecco’s phosphate buffered saline (PBS), molecular grade water, and saponin detergent were obtained from EMD Millipore (Burlington, MA, United States). Tris-EDTA, nuclease-free water was obtained from VWR (Radnor, PA, United States). UltraCruz Blocking Agent and antibodies directed against CD63 (clone MX-49.129.5), TSG101 (clone c-2), and glypican-1 (GPC-1; clone 4D1) were obtained from Santa Cruz Biotechnology (Dallas, TX, United States). Directly conjugated anti-programmed death ligand-1 antibody (PD-L1; clone 2746) was obtained from Biotium (Fremont, CA, United States). Fluorophore conjugated, affinity-purified F(ab)₂ goat anti-mouse IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, United States). K₂EDTA plasma used for spiked gDNA and EV experiments was purchased from Innovative Research (Novi, MI, United States). Quantitative multiplex reference standard gDNA (HD701) was purchased from Horizon Discovery (Cambridge, United Kingdom). Cerebrospinal fluid was obtained from Gemini Bio-Products (West Sacramento, CA, United States).

Exosomes were isolated from HEK293TN cells expressing XPack™ constructs using Total Exosome Isolation (Life Technologies, Carlsbad, CA, USA; 4478359) reagents following the manufacturer’s instructions. The isolated exosome pellets were resuspended in 400 μL sterilized 1× PBS buffer. The concentrated exosomes were diluted 100-fold using sterilized PBS for further biophysical characterization using ZetaView, Nanoparticle Tracking Analysis (NTA) instrument (Particle Metrix) for measuring particle size and concentration. For in vitro labeling of exosomal RNA, 1 μL of SYTO^® RNASelect™ Green Fluorescent Cell Stain (ThermoFisher, Waltham, MA, USA; S32703) was added to the diluted exosome samples and incubated at 37 °C for 30 min. For determining uptake of the labeled exosomes by recipient cells, 2 × 10⁵ differentiated SH-SY5Y cells were seeded into a single well of 24 well-plate and cultured overnight. Fluorescently labeled exosomes were added to the cells and incubated at 37 °C for 24 h before analyzing by fluorescence microscopy.

Ultracentrifuged material from stimulated MSC cultures was assessed for exosome/miRNA presence using the SYTO RNASelect Green Fluorescent Cell Stain per vendor provided instructions (Thermo Fisher Scientific, Waltham MA). Processed material was mixed 1:1 with exosome free RPMI media and subsequently cultured with fresh from frozen PBMCs for 24 hours. Imaging was performed using an EVOS Imaging System (Thermo).

To examine MSC-EV uptake by target cells such as vascular endothelial cells in vitro, MSC-EV samples were stained with lipophilic Vibrant DiO Cell-Labeling Solution (ThermoFisher Scientific) and SYTO RNA Select green fluorescent cell stain (ThermoFisher Scientific) according to the manufacturer’s protocols to label membrane lipids and RNA within the vesicles, respectively. MSC-EVs were stained for 20 min in the dark at RT and freshly used for experiments.