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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Isolation protocol
  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV180042 1/1 Mus musculus Cell culture supernatant dUC Ikebuchi, Yuki 2018 55%

Study summary

Full title
All authors
Ikebuchi Y, Aoki S, Honma M, Hayashi M, Sugamori Y, Khan M, Kariya Y, Kato G, Tabata Y, Penninger JM, Udagawa N, Aoki K, Suzuki H.
Journal
Nature
Abstract
Receptor activator of nuclear factor-kappa B (RANK) ligand (RANKL) binds RANK on the surface of oste (show more...)Receptor activator of nuclear factor-kappa B (RANK) ligand (RANKL) binds RANK on the surface of osteoclast precursors to trigger osteoclastogenesis. Recent studies have indicated that osteocytic RANKL has an important role in osteoclastogenesis during bone remodelling; however, the role of osteoblastic RANKL remains unclear. Here we show that vesicular RANK, which is secreted from the maturing osteoclasts, binds osteoblastic RANKL and promotes bone formation by triggering RANKL reverse signalling, which activates Runt-related transcription factor 2 (Runx2). The proline-rich motif in the RANKL cytoplasmic tail is required for reverse signalling, and a RANKL(Pro29Ala) point mutation reduces activation of the reverse signalling pathway. The coupling of bone resorption and formation is disrupted in RANKL(Pro29Ala) mutant mice, indicating that osteoblastic RANKL functions as a coupling signal acceptor that recognizes vesicular RANK. RANKL reverse signalling is therefore a potential pharmacological target for avoiding the reduced bone formation associated with inhibition of osteoclastogenesis. (hide)
EV-METRIC
55% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
209.7 (pelleting) / 209.7 (washing)
Protein markers
EV: CD9/ CD63/ CD81/ RANK
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
Function, Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
RANKL-stimulated
EV-producing cells
RAW264.7,mouse primary osteoclasts
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Cell viability
50
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
209.7
Wash: time (min)
60
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
209.7
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Concentration
2.5-3.5
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, CD81
Not detected contaminants
Calnexin
ELISA
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Proteomics database
Yes
Fluorescent NTA
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Close-up
Extra information
Antibody details for Western blot: rabbit anti-CD9 (Abcam, CatNo. ab92726, clone EPR2949, LotNo. GR260186-10, 1:1000), rat anti-CD63 (MBL, CatNo. D263-3, clone R5G2, LotNo. 014, 1:500), hamster anti-CD81 (Bio-Rad, CatNo. MCA1846GA, clone Eat2, LotNo. 0515, 1:500), rabbit anti-Calnexin (Abcam, CatNo. ab22595, polyclonal, LotNo. GR86850-1, 1:500)
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