Oct 02, 2025
Establishment and culture of mouse oviductal organoids and isolation and characterization of their secreted extracellular vesicles_Supporting Information File S1
Peer-reviewed method
- Riley Thompson1,
- Mindy A Meyers1,
- Richard McCosh1,
- Fiona K Hollinshead1
- 1Colorado State University
- PLOS ONE Lab ProtocolsTech. support email: [email protected]
External link: https://doi.org/10.1371/journal.pone.0337587
Protocol Citation: Riley Thompson, Mindy A Meyers, Richard McCosh, Fiona K Hollinshead 2025. Establishment and culture of mouse oviductal organoids and isolation and characterization of their secreted extracellular vesicles_Supporting Information File S1. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5kzz4v1b/v1
Manuscript citation:
Thompson-Brandhagen RE, Meyers MA, McCosh RB, Hollinshead FK (2025) Establishment and culture of mouse oviductal organoids and isolation and characterization of their secreted extracellular vesicles. PLOS One 20(12). doi: 10.1371/journal.pone.0337587
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 16, 2025
Last Modified: October 02, 2025
Protocol Integer ID: 223005
Keywords: Isoflurane gas, Ovariectomy, Organoid culture, isolation of extracellular vesicle, extracellular vesicle, characterization of extracellular vesicle, oviductal organoid, isolation of oviductal cell, oviductal cell, establishment of organoid, vesicles-supporting information file s1, collection of mouse oviduct, organoids for downstream application, collecting organoid, organoid, mouse oviduct, culture of mouse, cell
Abstract
This protocol details the description of supporting information file S1 which includes preparation and collection of mouse oviducts, isolation of oviductal cells and establishment of organoids, passaging of organoids, collecting organoids for downstream applications, isolation of extracellular vesicles from spent culture medium, and characterization of extracellular vesicles from spent culture medium.
Materials
Equipment:
- Anesthesia machine (VetEquip #901807)
Equipment
Anesthesia machine
NAME
VetEquip
BRAND
901807
SKU
LINK
- Anesthesia induction chamber (VetEquip #941444)
Equipment
Anesthesia induction chamber
NAME
VetEquip
BRAND
941444
SKU
LINK
- Intraoperative mouse heating pad (Stoelting #53850M)
Equipment
Intraoperative mouse heating pad
NAME
Stoelting
BRAND
53850M
SKU
LINK
- Postoperative cage warmer (Fisher #NC1867292)
Equipment
Postoperative cage warmer (Fisher #NC1867292)
NAME
Fisherbrand
BRAND
NC1867292
SKU
LINK
- Clippers (Kent Scientific #CL7300-KIT)
Equipment
Clippers (Kent Scientific #CL7300-KIT)
NAME
Kent Scientific
BRAND
CL7300-KIT
SKU
LINK
- Surgical scissors (VWR #63042-002)
Equipment
Surgical scissors
NAME
VWR
BRAND
63042-002
SKU
LINK
- Thumb forceps (VWR #10198-012)
Equipment
Thumb forceps
NAME
VWR
BRAND
10198-012
SKU
LINK
- Dissection microscope (Euromex #NZ.1903-B)
- Micro-scissors (Ambler Surgical #79-582)
- Biosafety cabinet (Labconco #302411101)
Equipment
Biosafety cabinet
NAME
Labconco
BRAND
302411101
SKU
LINK
- 20, 200, and 1000 μL Pipettors (ThermoScientific #4641180N, 4641210N, 4641230N)
Equipment
20, 200, and 1000 μL Pipettors
NAME
ThermoScientific
BRAND
4641180N
SKU
LINK
- Microcentrifuge (ThermoScientific #75002447)
Equipment
Microcentrifuge
NAME
ThermoScientific
BRAND
75002447
SKU
LINK
- Inverted microscope (Nikon TMS)
- CO2 incubator (ThermoScientific #51030285)
- Ultracentrifuge (Beckman Coulter Optima XE-90)
- Nanoparticle tracking analyzer (NTA; Particle Metrix ZetaView QUATT)
- Jess ProteinSimple western blot instrument (BioTechne #004-650)
Equipment
Jess ProteinSimple western blot instrument
NAME
BioTec
BRAND
004-650
SKU
LINK
Materials:
- Fluriso Isoflurane liquid (MWI #502017)
- Medical grade oxygen (Airgas OX USPEAMRI)
- VaporGuard Activated Charcoal filter (VWR #89012-608)
- Buprenorphine Base Lab in Polymer Injection Solution (Wedgewood Connect 0.5 mg/mL)
- 4-0 monofilament suture with reverse cutting needle (Vet One #V1-601074)
- Wound clips (Fine Science Tools #12040-01)
Equipment
Wound clips
NAME
Fine Science Tools
BRAND
12040-01
SKU
LINK
- Betadine (MWI #001574)
- Sterile surgical gloves (MWI #064374)
- No. 10 scalpel blades (VWR #76457-442)
- 35 × 10 mm tissue culture dishes (Corning #353001)
Equipment
35 × 10 mm tissue culture dishes
NAME
Corning
BRAND
353001
SKU
LINK
- 48-well culture plates (Corning #3548)
Equipment
48-well culture plates
NAME
Corning
BRAND
3548
SKU
LINK
- 20, 200, and 1000 μL pipette tips (ThermoScientific #94420218, 94420318, and 94420813)
Equipment
ClipTip™ Filtered Pipette Tips
NAME
Thermo Scientific
BRAND
94420813
SKU
LINK
- Microcentrifuge tubes (VWR #490004-444)
Equipment
Microcentrifuge tubes
NAME
VWR
BRAND
490004-444
SKU
LINK
- Ultracentrifuge tubes (Seton Scientific #7022)
- 20 mL syringes (MWI Animal Health #670063)
- 0.22 μm syringe filters (Cell Treat #229747)
Equipment
0.22 μm syringe filters
NAME
Cell Treat
BRAND
229747
SKU
LINK
- Cultrex UltiMatrix phenol red-free, growth factor reduced (Biotechne #BME001-05)
Equipment
Cultrex UltiMatrix phenol red-free, growth factor reduced (Biotechne #BME001-05)
NAME
Biotechne
BRAND
BME001-05
SKU
LINK
- Phosphate buffered saline (PBS; Accuris Life Science Reagents #EB1200)
Equipment
Phosphate buffered saline (PBS; Accuris Life Science Reagents #EB1200)
NAME
Accuris Life Sciences
BRAND
EB1200
SKU
LINK
- EV-free PBS (EMD Millipore #TMS-012-A)
Equipment
EV-free PBS
NAME
Sigma-Aldrich
BRAND
TMS-012-A
SKU
LINK
- Organoid Harvesting Solution (Biotechne #3700-100-01)
Organoid Harvesting SolutionBio-TechneCatalog #3700-100-01
- 4% paraformaldehyde (ThermoFisher #J19943-K2)
- 2% agarose prepared with water (Bio-Rad #1613101)
2% agarose prepared with waterBio-Rad LaboratoriesCatalog #1613101
- 70% ethanol
- 25% glutaraldehyde (Electron Microscopy Sciences #16220)
25% glutaraldehydeElectron Microscopy SciencesCatalog #16220
- Sodium cacodylate trihydrate (Electron Microscopy Sciences #12310)
Sodium cacodylate trihydrateElectron Microscopy SciencesCatalog #12310
- Sucrose
- Deionized water
- DMEM/F12 (Gibco #21041-025)
DMEM/F12 (Gibco #21041-025)Gibco - Thermo Fisher ScientificCatalog #21041-025
- Parafilm (Millipore Sigma #P6543)
Parafilm (Millipore Sigma #P6543)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P6543
- Formvar/carbon supported copper grids (Millipore Sigma #930261)
Equipment
Formvar/carbon supported copper grids
NAME
Millipore Sigma
BRAND
930261
SKU
LINK
- 2% uranyl acetate (Electron Microscopy Sciences #224002)
- CD9 primary antibody (abcam #ab307085)
CD9 primary antibodyAbcamCatalog #ab307085
- Hsp70 primary antibody (System Biosciences #EXOAB-Hsp70A-1)
- CYCS primary antibody (Sino Biological #102139-T42)
CYCS primary antibodySino BiologicalCatalog #102139-T42
- Goat anti-rabbit secondary antibody (Biotechne #042-206)
Goat anti-rabbit secondary antibodyBio-TechneCatalog #042-206
- Reagents for Jess western blot (see manufacturer guidelines)
- RIPA Buffer (Sigma-Aldrich #R0278)
RIPA BufferMerck MilliporeSigma (Sigma-Aldrich)Catalog #R0278
- Protease Inhibitor Cocktail Tablets (Roche #11836170001)
Protease Inhibitor Cocktail TabletsRocheCatalog #11836170001
- Antibody Diluent 2 (Protein Simple #042-203)
Antibody Diluent 2Bio-TechneCatalog #042-203
- Luminol-S (Protein Simple #043-311)
- Peroxide (Protein Simple #043-379)
- Streptavidin HRP (Protein Simple #042-414)
- EZ Standard Pack (Protein Simple #PS-ST01EZ)
- Protein Normalization Reagent (Protein Simple #043-824)
- Protein Normalization Reconstitution Agent (Protein Simple #043-823)
- 12-230 kDA Fluorescence Separation 8x25 Capillary Cartridges (Protein Simple #SM-FL004-1)
Handling medium:
| A | B | C | D | |
| Component | Manufacturer | Catalog No. | Concentration | |
| MEM Eagle with Earle’s Salts | Sigma | M2279 | N/A | |
| HEPES | Sigma | H0887 | 25 mM | |
| Penicillin/Streptomycin | Sigma | P4333 | 1% | |
| Sodium pyruvate | Gibco | 11360-070 | 0.1 mM | |
| Glutamax | Gibco | 35050-061 | 2 mM | |
| Fetal bovine serum (FBS) | Peak Serum | PS-FB1 | 20% v/v |
MEM Eagle with Earle’s SaltsMerck MilliporeSigma (Sigma-Aldrich)Catalog #M2279
HEPES solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #H0887
Penicillin-StreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #P4333
Sodium PyruvateGibco - Thermo Fisher ScientificCatalog #11360070
GlutamaxGibco - Thermo Fisher ScientificCatalog #35050-061
Fetal Bovine Serum (FBS) - US OriginPeak Serum, IncCatalog #PS-FB1
Organoid medium:
| A | B | C | D | |
| Component | Manufacturer | Catalog No. | Concentration | |
| DMEM/F12 without phenol red with L-glutamine | Gibco | 21041-025 | N/A | |
| Penicillin/Streptomycin | Sigma | P4333 | 1% | |
| B27 Plus | Gibco | A35828-01 | 2% | |
| N2 | Gibco | 17502-048 | 1% | |
| Insulin-transferrin-selenium | Gibco | 41400-045 | 1% | |
| Nicotinamide | Sigma | N0636 | 1 mM | |
| Recombinant human EGF | R&D Systems | 236-EG | 50 ng/mL | |
| Recombinant human FGF-10 | PeproTech | 100-26 | 50 ng/mL | |
| Recombinant human noggin | R&D systems | 6057-NG/CF | 100 ng/mL | |
| TGFβ/Alk inhibitor A83-01 | Tocris | 2939 | 0.5 μM | |
| N-acetyl-L-cysteine | EMD Millipore | 106425 | 1.25 mM | |
| SB202190 | Sigma | S7067 | 10 μM | |
| Y27632 | EMD Millipore | 688000 | 10 μM |
DMEM/F12 (Gibco #21041-025)Gibco - Thermo Fisher ScientificCatalog #21041-025
Penicillin-StreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #P4333
B-27™ Plus Supplement (50X)Gibco - Thermo Fisher ScientificCatalog #A3582801
N2 supplementGibco - Thermo Fisher ScientificCatalog #17502048
Insulin-Transferrin-Selenium (ITS -G) (100X)Thermo FisherCatalog #41400045
NicotinamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #N0636
Recombinant Human EGF Protein CFR&D SystemsCatalog #236-EG
Recombinant human nogginR&D SystemsCatalog #6057-NG/CF
TGFβ/Alk inhibitor A83-01TocrisCatalog #2939
N-acetyl-L-cysteineMerck Millipore (EMD Millipore)Catalog #106425
SB202190Merck MilliporeSigma (Sigma-Aldrich)Catalog #S7067
Y27632Merck Millipore (EMD Millipore)Catalog #688000
Preparation
Seek approval for use of research animals from local institution prior to start.
Ensure that mouse surgery is conducted in a clean space with adequate ventilation and that charcoal waste gas scavengers are used.
Collect mouse oviducts
Induce mouse for surgery using isoflurane gas (up to 5% isoflurane and medical grade oxygen at 3 liters per minute) in an induction chamber.
Once mouse is non-reactive, clip fur on dorsal body between shoulders and hips.
Position the mouse on a clean surface on top of an intraoperative rodent warmer with the nose of the mouse in a nose cone for inhalant anesthetic (1-5% isoflurane in medical grade oxygen) with periodic monitoring for depth of anesthesia.
Apply eye lubricant.
Administer buprenorphine (0.012 µL -0.016 µL of body weight) as an analgesic.
Scrub skin with povidone-iodine followed by 70% ethanol three times in the clipped surface.
About halfway between the bottom of the rib cage and the hip on the lateral surface of the dorsum, lift the skin with thumb forceps and cut a small incision with surgical scissors.
Lift the body wall with thumb forceps, then use surgical scissors to make a small incision into the abdominal cavity.
Beneath these incisions, exteriorize the fat pad containing the ovary using thumb forceps.
Clamp with hemostatic forceps between the ovary/oviduct and the uterus.
Use scissors or scalpel blade to excise the ovary/oviduct. Transfer ovary/oviduct to petri dish containing
1 mL Handling Medium.
Release hemostatic forceps while continuing to hold uterine stump with thumb forceps to ensure no bleeding from the uterine stump before releasing tissue back into abdominal cavity.
Use suture in a simple interrupted pattern to close the body wall.
Close skin with 1 or 2 wound clips.
Repeat procedure for opposite ovary.
Following bilateral ovariectomy, remove animal from isoflurane and recover in clean cage on a cage warmer. Monitor animals for pain and/or infection over next 7 to 10 days. Remove wound clips 7-10 days post-surgery.
Isolate mouse oviducts using dissection microscope
Remove adipose tissue that surrounds mouse ovary and oviduct using thumb forceps and micro-scissors.
Dissect the ovary away from the oviduct using micro-scissors or a scalpel blade.
Using gentle traction, extend the oviduct from a coiled to straight orientation.
Isolate oviductal cells and establish organoids
55m
TIP: Before starting, place UltiMatrix in 4 °C overnight to thaw and place a 48-well plate in 37 °C to warm.
Transfer the isolated oviduct to 1 mL Handling Medium in a new small tissue culture dish.
Continuing to use the dissection microscope, use micro-scissors and/or scalpel blade to expose the oviductal lumen.
Gently scrape the oviductal lumen to release the cells into the handling medium.
TIP: If too aggressive, oviduct will tear into small pieces that are more difficult to scrape.
Note
ALTERNATIVE OPTION: Co-incubate 1-2 mm sections of isolated oviduct with
collagenase to enzymatically isolate the cells.
Remove large tissue pieces from the Handling Medium containing the scraped cells.
Transfer the cell solution to a microcentrifuge tube.
Centrifuge at 600 x g for 00:10:00 at Room temperature .
10m
Remove the supernatant.
TIP: Cell pellet may be too small to see but is likely adhered to the bottom/side of the tube. Slowly remove as much supernatant as possible.
Add 20X cold UltiMatrix to estimated cell pellet size.
- TIP: If cell pellet cannot be visualized, add 50 µL UltiMatrix.
- TIP: Leave UltiMatrix in 4 °C as long as possible and place on ice when not in 4 °C . Quickly mix the cells with UltiMatrix while avoiding bubbles (bubbles do not appear to affect organoid growth but make imaging more difficult).
Place 25 µL droplets of the cells in UltiMatrix in the center of each well in a pre-warmed 48-well plate.
Place plate in 37 °C for 00:30:00 -00:45:00 to allow UltiMatrix droplets to become firmer.
45m
Add 250 µL warm (37 °C ) organoid culture medium to the wells containing the droplets.
TIP: Slowly run the culture medium down the side of the well rather than on top of the droplet.
Add 1 mL PBS to surrounding wells in the plate to reduce evaporation of the culture medium.
Replace plate in 37 °C , 5% CO2 incubator.
Remove and replace half of the organoid culture medium every two to three days.
- TIP: Some evaporation will occur-- aim to leave 125 µL of the culture medium in each well. Then add 125 µL of fresh organoid culture medium down the side of the well.
- TIP: Orient pipette tip toward the edge of the well because the droplet containing cells should not be present in that location. Remove spent medium slowly to prevent disruption of the droplet.
Note
For a more uniform number of cells per well, particularly if assessing organoid growth rates, dissociate the organoids into single cells using warm TrypLE Express Enzyme for 00:20:00 with intermittent pipetting. Then count the cells on a hemocytometer using trypan blue 1:1 with the cell solution aliquot. After calculating the live cell concentration, add UltiMatrix to the cell pellet at a ratio that will result in 5,000 or 10,000 cells per well in 25 µL droplets.
Passaging organoids
1h 55m
Timing of passage should occur when the organoids are the appropriate size and coloration, which is approximately every 7 days.
TIP: Organoids should be large enough to appreciate a lumen but should be a bright, light color. For mouse oviductal organoids, a dark color is indicative of cellular degeneration.
To passage, transfer the contents of each well to a microcentrifuge tube by scraping and removing with a P1000 pipette.
Centrifuge at 600 x g for 00:10:00 at Room temperature .
10m
Remove supernatant.
TIP: UltiMatrix will be pelleted with the cells. Try not to remove at this time since cells will be removed with the UltiMatrix.
Add 200 µL DMEM/F12.
Using a P200 pipette set at 150 µL , pipette 500X. Avoid producing bubbles.
Add 800 µL DMEM/F12.
Centrifuge at 600 x g for 00:10:00 at Room temperature .
10m
Remove supernatant.
- TIP: At this point, cells will be in a distinct separate pellet layered below the UltiMatrix. Attempt to remove as much UltiMatrix as possible without disturbing the cell pellet.
- TIP: Can increase centrifugation speed and time to 1000 x g for 00:10:00 if cell pellet is not forming well.
10m
Add 200 µL DMEM/F12.
Using a P200 pipette set at 150 µL , pipette 300X. Avoid producing bubbles.
Add 800 µL DMEM/F12.
Centrifuge at 600 x g for 00:10:00 at Room temperature .
10m
Remove supernatant.
Add 20X cold UltiMatrix to pellet and mix using pipette.
- TIP: Estimate the pellet size and add 20 times more UltiMatrix to the cell pellet. Avoid bubbles.
- TIP: Hold microcentrifuge tube toward the top so that hand is not warming the UltiMatrix.
Place 25 µL droplets of the cells in UltiMatrix in the center of each well in a pre-warmed 48-well plate.
Place plate in 37 °C for 00:30:00 -00:45:00 to allow UltiMatrix droplets to become less fluid.
1h 15m
Add 250 µL warm (37 °C ) organoid culture medium to the wells containing droplets.
TIP: Slowly run the culture medium down the side of the well rather than on top of the droplet.
Add 1 mL PBS to surrounding wells in the plate to reduce evaporation of the culture medium.
Replace plate in 37 °C , 5% CO2 incubator.
Remove and replace half of the organoid medium every two to three days.
Collecting organoids for downstream applications
7h 10m
Flash freeze for RT-qPCR.
Transfer the contents of each well to a microcentrifuge tube by scraping and removing with a P1000 pipette.
Centrifuge at 600 x g for 00:10:00 at Room temperature .
10m
Remove supernatant.
Add 250 µL cold Organoid Harvesting Solution per each well of organoids that was collected, and place On ice for 01:00:00 .
1h
Centrifuge at 600 x g for 00:10:00 at 4 °C .
10m
Remove supernatant.
Add 1 mL DMEM/F12 and resuspend cell pellet.
Centrifuge at 600 x g for 00:10:00 at 4 °C .
10m
Remove supernatant.
Plunge microcentrifuge tube containing cell pellet into liquid nitrogen and transfer to sample box for storage in -80 °C until use for RT-qPCR.
Fixation for histology.
Transfer the contents of each well to a microcentrifuge tube by scraping and removing with a P1000 pipette
Centrifuge at 600 x g for 00:10:00 at Room temperature .
10m
Remove supernatant.
Add 250 µL cold Organoid Harvesting Solution per each well of organoids that was collected, and place On ice for 01:00:00 .
1h
Centrifuge at 600 x g for 00:10:00 at 4 °C .
10m
Remove supernatant.
Add 1 mL PBS and resuspend cell pellet.
Centrifuge at 600 x g for 00:10:00 at 4 °C .
10m
Remove supernatant.
Add 500 µL 4% paraformaldehyde to tube without disturbing cell pellet. Incubate for 00:30:00 at Room temperature .
TIP: Run the paraformaldehyde slowly down the side of the tube.
30m
Remove the paraformaldehyde without disturbing cell pellet.
Add 50 µL warm 2% agarose to cell pellet, and transfer the agarose droplet containing the organoids with a P200 pipette with the tip cut off to a petri dish to cool.
TIP: Work quickly or the agarose will cool in the pipette tip.
After the agarose droplet containing organoids has cooled, transfer to a microcentrifuge tube containing 70% ethanol for storage in 4 °C until use for histology.
Fixation for transmission electron microscopy (TEM).
Transfer the contents of each well to a microcentrifuge tube by scraping and removing with a P1000 pipette.
Centrifuge at 600 x g for 00:10:00 at Room temperature .
10m
Remove supernatant.
Add 250 µL cold Organoid Harvesting Solution per each well of organoids that was collected, and place On ice for 01:00:00 .
1h
Centrifuge at 600 x g for 00:10:00 at 4 °C .
10m
Remove supernatant.
Add 1 mL PBS and resuspend cell pellet.
Centrifuge at 600 x g for 00:10:00 at 4 °C .
10m
Remove supernatant.
Add 500 µL TEM fixative (2% glutaraldehyde with 5% sucrose in 0.1 Molarity (M) sodium cacodylate; pH=7.4) to tube without disturbing cell pellet. Incubate for 02:00:00 at Room temperature .
2h
Remove the fixative without disturbing cell pellet.
TIP: If the pellet is disturbed, slow centrifuge (200 x g for 00:10:00 ) can be used to
re-pellet the organoids without damaging them.
10m
Add 500 µL 0.1 Molarity (M) cacodylate buffer (pH=7.4) for storage in 4 °C until use for TEM.
Isolation of extracellular vesicles from spent culture medium
3h 10m
Collect conditioned medium from organoid cell cultures.
Centrifuge at 750 x g for 00:10:00 at Room temperature .
10m
Remove and save supernatant. Discard pellet.
TIP: Can store supernatant at 4 °C for up to one week before continuing with initial processing.
Centrifuge at 3000 x g for 00:10:00 at 4 °C .
10m
Remove and save supernatant. Discard pellet.
Centrifuge at 17200 x g for 00:30:00 at 4 °C .
30m
Remove and save supernatant. Discard pellet.
Filter supernatant through a 0.22 μm syringe filter.
TIP: At this stage, can store in -80 °C before continuing EV isolation.
Ultracentrifuge samples at 120000 x g for 01:10:00 at 4 °C .
1h 10m
Remove and discard supernatant. Leave ~0.5 mL pellet.
Note
EV “pellets” often are not visible-be careful when removing supernatants.
Add 5 mL EV-free PBS to pellet and mix.
Ultracentrifuge samples at 120000 x g for 01:10:00 at 4 °C .
1h 10m
Remove and discard supernatant. Leave ~0.5 mL pellet, and mix vigorously to ensure EVs are not remaining in pellet stuck to tube.
Store the isolated EVs in -80 °C until characterization.
Characterization of extracellular vesicles from spent culture medium
5m
Nanoparticle Tracking Analysis of EVs.
Thaw isolated EVs, if frozen.
Dilute an aliquot of EVs until ZetaView NTA reads an average particle count of 50 to 500 particles per frame.
Capture data at 11 positions with instrument in scatter mode.
Note the particle size distribution and concentration after all data points are captured—be sure to calculate the concentration based on the dilution factor.
Note
1:200 is a good starting EV dilution for NTA.
Preparation of EVs for TEM.
Thaw isolated EVs, if frozen.
Transfer 30 µL of purified EVs onto parafilm.
Cover the EV droplets with formvar/carbon supported copper grids. Let incubate at Room temperature for 00:05:00 to allow the grid to absorb the EVs.
5m
Wash the grid with drops of ddH2O.
Add 30 µL of 2% uranyl acetate to fix.
Evaluate the EVs on the grid using a transmission electron microscope.
Jess Simple western blot for EVs.
Prepare each EV sample by combining 3 µL EVs and 1 µL RIPA RTU (1 tablet of protease inhibitor + 1 mL RIPA buffer) per antibody to be evaluated.
Prepare antibody dilutions. Dilute CD9 antibody 1:25. Dilute Hsp70 antibody 1:50. Dilute CYCS antibody 1:50.
Use manufacturer protocol to complete Jess Simple Western Blot.