Format

Send to

Choose Destination
Methods. 2015 Oct 1;87:64-74. doi: 10.1016/j.ymeth.2015.03.028. Epub 2015 Apr 3.

Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis.

Author information

1
Nuffield Department of Obstetrics & Gynaecology, University of Oxford, Womens Centre Level 3, John Radcliffe Hospital, Oxford OX3 9DU, UK. Electronic address: rebecca.dragovic@obs-gyn.ox.ac.uk.
2
Nuffield Department of Obstetrics & Gynaecology, University of Oxford, Womens Centre Level 3, John Radcliffe Hospital, Oxford OX3 9DU, UK. Electronic address: gavin.collett@obs-gyn.ox.ac.uk.
3
Malvern Instruments, London Road, Minton Park, Amesbury SP4 7RT, UK. Electronic address: Patrick.Hole@malvern.com.
4
Nuffield Department of Clinical Laboratory Science, University of Oxford, John Radcliffe Hospital, OX3 9DU Oxford, UK. Electronic address: david.ferguson@ndcls.ox.ac.uk.
5
Nuffield Department of Obstetrics & Gynaecology, University of Oxford, Womens Centre Level 3, John Radcliffe Hospital, Oxford OX3 9DU, UK. Electronic address: christopher.redman@obs-gyn.ox.ac.uk.
6
Nuffield Department of Obstetrics & Gynaecology, University of Oxford, Womens Centre Level 3, John Radcliffe Hospital, Oxford OX3 9DU, UK. Electronic address: ian.sargent@obs-gyn.ox.ac.uk.
7
Nuffield Department of Obstetrics & Gynaecology, University of Oxford, Womens Centre Level 3, John Radcliffe Hospital, Oxford OX3 9DU, UK. Electronic address: dionne.tannetta@obs-gyn.ox.ac.uk.

Abstract

The human placenta releases multiple types and sizes of syncytiotrophoblast (STB) extracellular vesicles (EV) into the maternal circulation that exhibit diverse biological activities. The placental perfusion technique enables isolation of these STBEV, but conventional flow cytometry can only be used to phenotype EV down to ∼300 nm in size. Fluorescence Nanoparticle Tracking Analysis (fl-NTA) has the potential to phenotype EV down to ∼50 nm, thereby improving current characterisation techniques. The aims of this study were to prepare microvesicle and exosome enriched fractions from human placental perfusate (n=8) and improve fl-NTA STBEV detection. Differential centrifugation and filtration effectively removed contaminating red blood cells from fresh placental perfusates and pelleted a STB microvesicle (STBMV) fraction (10,000×g pellet - 10KP; NTA modal size 395±12 nm), enriched for the STB marker placental alkaline phosphatase (PLAP) and a STB exosome (STBEX) fraction (150,000×g pellet - 150KP; NTA modal size 147±6 nm), enriched for PLAP and exosome markers Alix and CD63. The PLAP positivity of 'standard' 10KP and 150KP pools (four samples/pool), determined by immunobead depletion, was used to optimise fl-NTA camera settings. Individual 10KP and 150KP samples (n=8) were 54.5±5.7% (range 17.8-66.9%) and 30.6±5.6% (range 3.3-51.7%) PLAP positive, respectively. We have developed a reliable method for enriching STBMV and STBEX from placental perfusate. We also standardised fl-NTA settings and improved measurement of PLAP positive EV in STBMV. However, fl-NTA is not as sensitive as anti-PLAP Dynabead capture for STBEX detection, possibly due to STBEX having lower surface expression of PLAP. These important developments will facilitate more detailed studies of the role of STBMV and STBEX in normal and pathological pregnancies.

KEYWORDS:

Exosomes; Extracellular vesicles; Flow cytometry; Fluorescence NTA; Microvesicles; Placenta perfusion

PMID:
25843788
PMCID:
PMC4641874
DOI:
10.1016/j.ymeth.2015.03.028
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center