title: Effect of storage on physical and functional properties of extracellular vesicles derived from neutrophilic granulocytes.
abstract: OBJECTIVE
  To carry out a systematic study on the effect of different storage conditions on the number as well as the physical and functional properties of antibacterial extracellular vesicles (EVs) derived from human neutrophilic granulocytes.
  
  
  METHODS
  Production of EVs with antibacterial properties was initiated by opsonized Zymosan A particles. The number of released fluorescent EVs was determined by flow cytometry following careful calibration. Physical properties and size of EVs were investigated by flow cytometry, dynamic light scattering and electron microscopy. Functional properties of EVs were tested by bacterial survival assay.
  
  
  RESULTS
  Storage at +20°C or +4°C resulted in a significant decrease of EV number and antibacterial effect after 1 day. Storage at -20°C did not influence the EV number up to 28 days, but induced a shift in EV size and almost complete loss of antibacterial function by 28 days. Storage at -80°C had no significant effect either on EV number or size and allowed partial preservation of the antibacterial function up to 28 days. Snap-freezing did not improve the results, whereas the widely used cryoprotectants induced EV lysis.
  
  
  CONCLUSIONS
  Storage significantly alters both the physical and functional properties of EVs even if the number of EVs stays constant. If storage is needed, EVs should be kept at -80°C, preferably not longer than 7 days. For functional tests, freshly prepared EVs are recommended.
authors.affiliation: Department of Physiology, Semmelweis University, Budapest, Hungary.
authors.forename: Ákos M
authors.initials: ÁM
authors.lastname: Lőrincz
authors.affiliation: Department of Physiology, Semmelweis University, Budapest, Hungary.
authors.forename: Csaba I
authors.initials: CI
authors.lastname: Timár
authors.affiliation: Department of Physiology, Semmelweis University, Budapest, Hungary.
authors.forename: Krisztina A
authors.initials: KA
authors.lastname: Marosvári
authors.affiliation: Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary.
authors.forename: Dániel S
authors.initials: DS
authors.lastname: Veres
authors.affiliation: Institute of Experimental Medicine of the Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Lilla
authors.initials: L
authors.lastname: Otrokocsi
authors.affiliation: Institute of Experimental Medicine of the Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Ágnes
authors.initials: Á
authors.lastname: Kittel
authors.affiliation: Department of Physiology, Semmelweis University, Budapest, Hungary; Ligeti@puskin.sote.hu; Ligeti.Erzsebet@med.semmelweis-univ.hu.
authors.forename: Erzsébet
authors.initials: E
authors.lastname: Ligeti
keywords: antibacterial effect; extracellular vesicle; microvesicle; storage conditions; vesicle function