Hi, can you give some more detail to your protocol. Because there are may posibilities to isolate Exosomes.

Thanks

hi, 

Do I understand it correctly that you capture exosome with beads (which beads?, size of the beads? magnetic?), then apply the bead-exosome complex on the grid surface? if thats the case it will be a major challenge (magnetic beads and magnetic lenses in the microscope). for more info it would be interesting to have a look at the protocol. thanks

ketil

Hi Luai,

like the two colleagues I need more detailed information about your protocol to answer the question. We did a whole lot of EM on exosomes, but it's important to know what steps in which order you perform.

Best,

Klaus

Thanks everyone for replying.

I isolate my exosome using ultra-centrifuge. After isolating them I place them on the grid for EM microscopy. When I just do the negative stain I get great pictures. When I'm trying to do immune-labeling on the grid with CD63, at some point all my exosomes are getting washed off. When I look under the EM microscope at the end of the process there are no exosome at all on the grid. I'm using the classic protocol for EM immuno labeling protocol.

Hi Luai,

this is a common issue. As exosomes are tiny structures, their adherence to the copper grids is not that strong, so they are washed away very easily.

At least your primary antibody should be added to the exosomes before ultracentrifugation. And the removal of every liquid (e.g. washing steps) from the grid should be performed very tenderly using a filter paper.

Last not least the adherence of such tiny structures is much better on carbon coated grids, though they are expensive.

All the best

Hi, 

I put together a few general things to consider (which will also apply for exosomes) when preparing for negative stain and TEM:

the first point here is critical!

• Glow discharge carbon coated copper grids to improve adsorption by making the surface hydrophilic. The drop of pre-enriched exosomes can be as small as 5-10 µL. Cover the grids during incubation with a lid to avoid any disturbance of the grid).
• Upconcentration of exosomes on the grids can be performed by incubating the grid on a drop of exosomes for 10 minutes, then remove the grid for a few minutes and then put it back on the same drop of exosomes again. The exosomes will stick to the water-air interface and can be “picked up” by the grid in several steps. 
• For double labeling different sizes of protein A gold are used such as 5 nm, 10 nm, 15 nm or 20 nm.
• In order to obtain the best possible contrast and resolution several different solutions should be tested. (Aqueous Uranyl Acetate: A 1% to 3% solution of uranyl acetate dissolved in water can be used to negatively stain of many samples. The stain has a low pH so this solution is not recommended for particles that are unstable in acid conditions, Neutral Phosphotungstic Acid: A 1% to 3% solution of phosphotungstic acid is made up in water and the pH is adjusted to 7 using sodium hydroxide. This is a useful stain for many samples but is especially good for viruses that dissociate at low pH. The stain produces less contrast than the uranyl acetate. Ammonium Molybdate: Make up a 1% solution of ammonium molybdate in water. This solution has also been used to negatively stain thawed, thin cryosections of fixed cells.
• Remove excess methyl cellulose and uranyl acetate by using a filter paper and place the grid perpendicular to the filter paper. Barely touch the grid to the filter paper and move the grid until no methyl cellulose is removed. It should only be a thin layer of methyl cellulose covering the exosomes on the grid.

I hope this will help.

kind regards

ketil

I will give the carbon grids a try. Lets see if this works! 

Thanks all 

I have to ask abiut methylcellulose preparation. Have I to ultracentrifuge methylcellulose as in the Thery protocol?

Hi, 

here is a protocol commonly used.

Methyl cellulose 2% (200ml)

1. heat 196 mL dH2O to a temp of 90 degrees C

2. Add 4g MC while stirring

3. rapidly cool the solution while stirring

4. seal with parafilm and stir O/N at 4 degrees C

5. Stop stirring, let the solution “ripen” for 2 days at 4 degrees C

6. Add dH2O  till 200ml

7. centrifuge for 1.5 h at 100.000 G

8. Pour the supernatant in tubes with a screw cap

9. Store at 4 degrees C in the dark (shelf life: 6-9 months)

kind regards

ketil

thats cool! the protocol comes from the Utrecht crew

For exosome isolation and analysis form milk we do not have an optimized protocol. However, I know that prof S Gabrielson has performed exosome analysis using our Dynabeads. So a combination the protocols used in those publications with ready made Dynabeads (CD9, CD63, CD81) for isolation or analysis should work. 

kwp

Exosomes captured with the Dynabeads can easily be labeled and then the exosome-bead complex are introduced into the flow cytometer for analysis. One of our recent method papers (can be downloaded from my profile) demonstrates how this can be performed and which factors are critical for success. 

kwp

@Bj Malla, yes that as well as the other ones. we have described different scenarios depending on the starting material - being either pre-enriched samples (e.g. UC or precipitated exosomes) or going directly into cell culture media. its both papers, book chapter and powerpoint presentation (delivered as webinars). In terms of the book chapter I need to send it to you privately. If thats of interest please get in touch ([email protected])

kind regards

ketil

Dear all, thank you for sharing such informations!!

One question: can one use Collodion-coated copper grids to adhere the microvesicles?

Best regards

Daniel

@Luai the same here! I did not see any gold labeling and neither exosomes....those I saw before....which is the secondary ab dilution? I used 1:150 GAM with 10nm gold particles....

@monica, I have used 1:100. still waiting to obtain some carbon based grids.

@Luai thanks. Finally I saw immunogold on exosomes following the ketil's suggestions. I put the grids for three times 10 min each on the exosome drops. in this trial I used GAM 1:40.

Hi I am using Poly ethylene glycol to isolate exosomes. I am having som problems with immuno EM. My exosomes appear really dark and i dont see any gold spots.

I use 10 ml of cell culture medium to collect exosmes . The concentration of PEG is 12%. When i do immuno EM i dont see gold staining of exosomes. I use CD9 & CD 81 as markers. I incubated the carbon coated grids on exosomes for 1 min (3 x). Wash with 1x PBS for 30 sec (3 x) and block with 1% BSA for 20 mins at RT. Incubate with CD9 or CD 81(1:50, 2 ul in 100 ul of blocking buffer) for 30 min at RT. wash the grids with 1x PBS for 30 sec (3X). Incubate the grids with secondary antibody (1:50, 2 ul in 100 ul of blocking buffer). wash the grids with 1x PBS for 30 sec (3x). Last wash was done in distilled water. Finally grids were stained on 2% uranyl acetate and were imaged.

Do you see exosomes without immunolabeling? with just a negative stain (1%UA). 10ml seems very small amount of media...i would go at least 100-200ml...

Also, I would double check with western blots first that your exsomes are positive for CD9 and CD81...

HI please find the attached picture. This is just negative stained. I have used PEG 8000 to isolated exosomes from 10 ml. I am not sure whether i am looking at exosomes or some random stuff.

i am trying to isolate exosomes from HEK293T cells. How cells would i require to get a decent population of exosomes to do EM and westernblots.

@manoj . Hard to tell. I would confirm with western blot for the markers as well.

I usually try to fill two  T-175 flasks....so 30ml each...i collect the media after 3 days and replace with new media and again after 3 days collect. that way you should have about 120ml of media. I think its a good place to start. I also would recommend, if possible, to use ultra-centrifugation  isolation method. 

Hi Luai

Thank you for the suggestions. Is there any protocol you follow for UC. I know there is lot of literature out there talking about different rotors and ultra centrifuges having different k factors require different timings for isolating exosomes. We have a microultracentrifuge from sorvall which has a K factor of 60.