For flow cytometry characterization, EVs were suspended in 0.22-μm-filtered PBS/EDTA, distributed in flow cytometry tubes (1 × 108 EVs/tube), and stained with 1 μM CFDA-SE (VybrantTM CFDA SE Cell Tracer Kit, Thermo Fisher Scientific) at RT to visualize intact vesicles. Since CFDA-SE diffuses within vesicles only at RT, a control at 4°C was also included. A mixture of fluorescent beads with a diameter ranging from 100 to 900 nm (Megamix-Plus FSC and Megamix-Plus SSC, Biocytex, France) was used to discriminate EV size. Expression of typical EV markers, such as CD9 (APC Mouse Anti-Human CD9, 312108, BioLegend, San Diego, CA, United States), CD63 (PE-CF594 Mouse Anti-Human CD63, 565403, BD Biosciences), and CD81 (BV421 Mouse Anti-Human CD81, 740079, BD Biosciences), was evaluated within the CFDA-SE positive events. All the evaluations were performed using a CytoFLEX flow cytometer (Beckman Coulter), and data were analyzed using FlowJo. Dimensional analysis was performed by creating three dimensional gates (EVs <100 nm, 100 nm < EVs <160 nm, and 160 nm < EVs <900 nm).
Vybranttm cfda se cell tracer kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Vybrant™ CFDA SE Cell Tracer Kit is a reagent used for labeling and tracking cells in various applications. It contains 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFDA SE), a fluorescent dye that can be used to label living cells. This dye passively diffuses into cells and binds to intracellular proteins, allowing for the visualization and monitoring of cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Megamix plus fsc, by Biocytex (2 mentions)
Megamix plus ssc, by Biocytex (2 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Facsaria 2, by BD (1 mentions)
Periodic acid, by Merck Group (1 mentions)
Fluorescein isothiocyanate isomer 1 fitc, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Milli q water, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Quil a, by Brenntag (1 mentions)
3 protocols using vybranttm cfda se cell tracer kit
1
Characterizing Extracellular Vesicles by Flow Cytometry
2
Flow Cytometry Characterization of sEVs
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For flow cytometry characterization, sEVs were suspended in filtered PBS/EDTA, distributed in flow cytometry tubes (100 μl/tube) and stained with 1 μM CFDA-SE (VybrantTM CFDA SE Cell Tracer Kit, Thermo Fisher Scientific) at room temperature (RT) to visualize intact vesicles. Since CFDA-SE diffuses within vesicles only at RT, a control at 4 °C was also included. A mixture of fluorescent beads with a diameter from 100 to 900 nm (Megamix-Plus FSC and Megamix-Plus SSC, Biocytex, France) was used to discriminate sEV size. Expression of typical EV markers, such as CD9 (APC Mouse Anti-Human CD9, 312108, Biolegend, San Diego, CA, USA), CD63 (PE-CyTM7 Mouse Anti-Human CD63, 561982, BD Biosciences) and CD81 (BV421 Mouse Anti-Human CD81, 740079, BD Biosciences) was evaluated within the CFDA-SE positive events. All the evaluations were performed using a BD FACSAria II (BD Biosciences) and Flow Jo software for post-analyses.
3
Chitosan-based Vaccine Adjuvant Characterization
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Chitosan HMC + 70/5 (MW 81 kDa, DD 88 %), HMC + 85/5 (MW 18 kDa, DD 94 %) and HMC + 90/10 (MW 28 kDa, DD 97 %) were supplied by Heppe Medical Chitosan (HMC) GmbH (Halle, Germany). OVA (grade VII, from chicken egg white), phosphate buffer solution (PBS) tablets and fluorescein isothiocyanate (FITC -isomer I) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Quil-A was acquired from Brenntag Biosector (Frederikssund, Denmark). Fasted state simulated intestinal fluids (FaSSIF) were obtained from Biorelevant.com (London, UK), while phorbol myristate acetate (PMA) was from Sigma-Aldrich (München, Germany). Ethanol 96 % (EtOH) was acquired from VWR International (Darmstadt, Germany), and acetic acid (AA) >99%, stomach porcine mucin, periodic acid and Schiff's reagent for aldehydes were supplied from Sigma-Aldrich (Steinheim, Germany). RPMI 1640 was obtained from Gibco (Carlsbad, CA, USA), and VyBrant TM CFDA SE Cell Tracer Kit was from Thermo Fisher (Waltham, USA). Lipopolysaccharides (LPS) from Salmonella enterica serotype enteritidis and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Paraformaldehyde was obtained from Electron Microscopy Sciences (Hatfield, USA), whereas milliQ water (Millipore Corporation, Billerica, MA, USA) was freshly produced in all cases.
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