Lessons of the electron microscopic analysis of microvesicle preparations produced by routinely used protocols

rapidcrimsonMécanique

22 févr. 2014 (il y a 3 années et 1 mois)

59 vue(s)

Lessons of the electron microscopic analysis of microvesicle preparations produced by routinely
used protocols

Ágnes Kittel
1
, Tamás Géza Szabó
2
, Edit I Buzás
2

1
Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary

2
Department of Genetics, Cell
-

and Immunobiology, Semmelweis University, Budapest, Hungary


Although microvesicles (MVs) and their significance in the mechanisms of both intercellular
communication and in wide spectrum of biological functions has been docum
ented, the diverse
protocols applied for their isolation and detection make the comparison of the results difficult, and
in some cases even impossible.

Our goal was to demonstrate the effect of different isolation techniques on the composition of the
re
sulting MV
-
containing pellets.

Different, routinely used centrifugation forces (such as 15,000 g, 20,500g, 100,000g and 200,000g)
and centrifugation times (60 and 120 minutes) were compared in the case of MVs derived from the
BV2 microglial cell line. The

resulting pellets were analysed by transmission electron microscopy.

The different sedimentation protocols resulted in differentially damaged MV populations. The use of
200 000g during MV isolation was found to hold the risk of destroying the ultrastruct
ure of MVs.
Importantly, different parts of the same MV pellet showed highly different distribution of size, shape
and electron density of MVs.

Electron microscopy suggest
s

that differential centrifugation isolation of MVs (without filtration)
does not yi
eld in homogenous populations of vesicles, and draws our attention to the fact that by
selecting a given electron microscopic field of an MV pellet, we may introduce significant bias to the
assessment of MV preparations.

Taken together, our results sugges
t that there is a strong need for standardization of the electron
microscopic investigation of MVs.


Microvesicles, exosomes, reclosed membrane fragments and cell debris in the pellet of partially
purified supernatant of BV2 microglial cell line