Catching microparticles - a methodical comparison
C. Plattfaut, F. Gieseler, A. Freund, G. Riemekasten, A. Müller (1Lübeck, Germany)
Laboratory
Date: 16.02.2017,
Time: 08:00 - 09:15
Objective: The field of microparticle (MP) research is constantly expanding. In 2000 there were 155 publications about MPs, in 2015 pubmed lists 1174. The increasing number of research groups calls for standardization of MP isolation, to ensure comparability and transparency of results. In 2013 Lacroix R. et al published a protocol for standardization of sample preparation for flow cytometric analysis of microparticles (J Thromb Haemost 10.1111/jth.12207). We prepared our samples according to this protocol. For further isolation and accumulation of microparticles there is no common consensus. The majority of work groups uses high-speed- or ultra-centrifugation. In this study we compare two different methods for MP-accumulation: a) high-speed centrifugation of samples at 10.000g for 90minutes (2009_Muralidharan-Chari); b) capture with Annexin-V-coated magnetic beads (Gieseler et al. Cell Biol Int. 2013).
Methods: We analyzed patient derived effusion obtained from the University Hospital Schleswig-Holstein (UKSH, Germany). For depletion of larger cell debris we centrifuged our samples twice at 2.500g for 15 minutes and discarded the pellet, thus obtaining low speed centrifuged samples ("lsEV"). Afterwards we divided our samples, one part was centrifuged again at 10.000g for 90 minutes obtaining high-speed EVs ("hsEV"), the other part was processed according to our previously published capture method wher annexin-V coupled magnetic beads are used, obtaining captured EVs ("capEV"). Afterwards, we characterized the samples by high-resolution cytoflow as well as various ELISA based assays, and tested their cellular effects such as the activation of the PAR2/ G protein/ ERK signaling pathway and the induction of tumor cell migration.
Results: Soluble factors such as growth factors or cytokines, which might interfere in functional assays, are depleted by both assays. As compared to the ISTH protocol, both methods resulted in a depletion of small EVs such as exosomes. capEVs had higher tenase activity and they were more potent inducers of tumor cell migration than hsEV.
Conclusion: EV-capture by anexin-V coupled magnetic beads selects phosphatidylserine (PS) positive EVs, whereas high speed centrifugation includes all EVs, therefore the combination of the methods might be useful to determine different subtypes of EVs with different cellular effects.