Hi,

Paying attention to all steps in this workflow is important in order to get good signal in WB in the end. I would therefor suggest the following:

1. Optimization of cell culture conditions.

I am adding our in house system as an example. Cell culture and exosome production

culture adherent cells to confluency (RPMI 1640 w/ 10% FCS, 1 mM Sodium-pyruvat in cell culture bottles (225 cm2) in 37C w/ 5% CO2.

remove medium form confluent cells and add 50 mL of fresh medium.

After 3 days the cell-conditioned medium is removed and two centrifugation steps are performed on the pre conditioned medium prior to pre-enrichment (300xg for 10 minutes at 2-8oC and 2000xg for 30 minutes 2-8oC).

2. Test for exosome release using magnetic beads (Dynabeads)

depending on the cell type and their exosome production efficiency it is possible to pull out exosomes from the solution directly without any pre-enrichment step and perform e.g. flow analysis. This is very useful in order to check the status of the cells exosome release efficiency and find the correct time for exosome harvest. I would use Dynabeads CD9, Dynabeads, CD63 or Dynabeads CD81 (hopefully your exosomes do contain at least one of these common exosomal markers (could stain the host cell first for all these markers and do flow analysis – at least it will give you an indication).

A flow protocol may look like this:

Capture for flow

The protocol is based on isolation using 200.000 magnetic beads but it can be scaled up by increasing volumes and reagents proportionally. 

Preparation of pre-enriched exosome sample

Use cell culture directly (pre-enriched exosomes can also be used to test the pre-enriched exosome preparation).

Add Isolation Buffer to 100 µL final volume per 20 µL magnetic beads. 

Day 1.

Resuspend the magnetic beads by mixing for >10 minutes or vortexing for 30 s.

Transfer 20 µL magnetic beads into an appropriate tube.

Wash the magnetic beads by adding 200 µL of Isolation Buffer and mix well.

Place the tube on the magnet for 1 min and discard the supernatant.

Remove the tube from the magnet, and add pre-enriched exosome solution titrated with Isolation Buffer (100 µL final volume) to the magnetic beads. Mix well.

Incubate the tube ON (18-22 h) at 2-8°C with mixing.

Day 2.

Centrifuge the tube for 3-5 seconds to collect the sample at the bottom of the tube.

Wash the bead-bound exosomes by adding 300 µL of Isolation Buffer. Mix gently by pipetting.

Place the tube on the magnet for 1 min and discard the supernatant.

Remove the tube from the magnet and add 400 µL of Isolation Buffer. Mix gently by pipetting.

Place the tube on the magnet for 1 minute and discard the supernatant.

Resuspend the bead bound exosomes in 300 µL Isolation Buffer.

The bead bound exosomes are now ready to be stained for flow cyt.

Staining for flow cytometry

The staining antibody should be titrated for optimal signal to noise ratio, starting with the manufacturer`s recommended concentration for staining 1 x 106 cells.

Transfer desired staining antibodies to a flow tube.

Add 100 µL bead-bound exosomes to the tube. Mix gently by pipetting.

Incubate for 45-60 minutes at RT protected from light on a sample shaker (approximately 1000 rpm).

Wash the bead-bound exosomes by adding 300 µL of Isolation Buffer. Mix gently by pipetting (do not vortex).

Place the tube on the magnet for 1 minute and discard the supernatant.

Repeat the washing steps once and resuspend in the desired volume of Isolation Buffer for flow cytometry analysis

3. Direct approach or a pre-enrichment strategy.

The flow signal you will get might vary from very low/nothing or up to signals that are actually sufficient for further detailed analysis. Depending on this signal you may decide to continue to do direct isolation with the Dynabeads as they are compatible with many different downstream applications such as western, electron microscopy, MS, seq, PCR etc. If the signal you get from the pre-testing by e.g. flow is very low you may consider doing a pre-enrichment step (UC or precipitation).

A precipitation protocol can look like this:

• Harvest media.
• Centrifuge at 350xg for 10 minutes.
• Transfer the supernatant to a new tube.
• Centrifuge supernatant at 2000xg in 30 min at 2-8C.
• Mix 1 part cell-free culture media to 0.5 volumes of the Total Exosome
• Isolation (from cell culture media) reagent.
• Mix well by vortexing, or pipetting up and down until there is a homogenous solution.
• Incubate samples at 2-8C ON.
• Centrifuge the samples at 2-8C at 10.000xg for 1 hour (fixed angle rotor).’
• Discard supernatant. Exosomes are in the pellet at the bottom of the tube for swing-out rotors or at the tube-wall near the bottom for fixed angle rotor.
• To remove liquid and leave the tube upside down on a filter paper for 3-4 min
• Resuspend the pellet in a given volume in 1xPBS.
• Exosomes are ready for downstream analysis or further purification through affinity methods or aliquotation and freezing in -80°C 

4. Western blotting from pre-enriched exosomes

As the amount of protein can be low I try to add all starting material in each well to get a proper signal. Currently we are using gels which have the capacity to add 40 µL of sample (Bolt system). The suggestion below is scaled according to this well size.

A WB protocol might look like this:

• 24µl pre-enriched exosome sample
• 6µl 5xRIPA with prot inhib
• 10s sonication

→ incubate 15 min at 4°C

Exosome boiling

• 30µl of lysed exosomes
• 10µl 4xLDS  

→ incubate 10 min at 70°C

Apply all in one well

5. Western Blotting – subpopulations of exosomes isolated with magnetic beads

If initial flow signal is fine it is possible to isolate directly from cell culture media

If initial flow signal is very low pre-enrichment should be considered.

If one wants to obtain ultra-pure exosomes – Dynabeads decorated with prim Abs will allow to recover very clean population of exosomes, following initial purification e.g with Total Exosome Isolation (from cell media) or ultracentrifugation – or directly from culture media. These subpopulations of exosomes can be analysed by electron microscopy, flow cytomtery, WB and RNA contect etc.

To maximize the WB signal in cases with reduced amount of exosomes oe may consider increasing the amount of magnetic beads used for isolation. This will provide a larger surface for exosomes to dock onto in addition to improve the binding kinetics. Increasing the number of beads will give an increasing signal on WB. We have also been able to pull out exosomes directly from cell culture media without any pre-enrichment after optimizing the cell culture growth conditions and exosome harvest conditions followed by WB.

A protocol for exosomes isolated with magnetic beads may look like this (20 µL beads + 100 µL sample volume):

Lysis of exosomes on Dynabeads & Boiling of exosomes after bead isolation

• All Dynabeads (when supernatant is removed)
• Add 6µl of 5xRIPA with prot inh (1.25 µL 25x)
• Add 24 µL dH2O
• 10 s of sonication
• Incubate for 15 min on ice
• Remove beads by applying magnet10 µL 4xLDS
• 10 min at 70°C
• Add 40 µL/well (4-12% bis tris gels)

Maximize Blotting conditions:

For blotting I would recommend wet transfer to PVDF membranes.

Maximize signal:

I would strongly recommend to use the most sensitive substrate – in-house we have had great success with the Super Signal West Dura as this gives high signal intensity and duration and is suitable for using in combination CCD cameras.

CD63 detection:

CD63 can in itself be a challenge to detect. The Abs targeting tetraspanins often require non-reducing conditions during sample prep for WB – so no mercapto during boiling of your sample. Furthermore, the CD63 clone is also important. According to key opinion leaders in this field the CD63 should give a smear on WB between 30-60 kDa. The clone preferred is TS63 which we have used as well during development – it can also detect different smear patterns depending on the exosome origin – indicating that the glycosylation pattern is different depending on origin. Interestingly, the CD63 expression also seems to vary a great deal between different sources.

CD81 and CD9 detection:

CD81 and CD9 detection might be worth including as well as they give single bands around 30 kDa (but not all exosomes are e.g. CD9 positive and also the expression level differs between different sources). 

We have Abs that have been validated for western blot detection of CD9, CD63 and CD81

kind regards

ketil 

One can precipitate proteins (see link below) and reduce the volume to a desirable volume. The challenge would be to make proteins soluble again for the BCA. It is likely that some exosomal proteins will not be soluble at reduced volume of water.

Link: http://www.unr.edu/proteomics/sample-preparation/2d-protocols/protocol-chloroform-methanol

Its likely yield issue+PBS issue. Use 1x RIPA for resuspending exosomes (No PBS).

Try this kit below, which doesn't require ultracentrifugation.

https://www.thermofisher.com/order/catalog/product/4478359

Another possibility is to increase the amount of of starting material (conditioned media)

Here is a reference it might be useful:

https://www.ncbi.nlm.nih.gov/pubmed/15368299

Piero

Yes, maybe the easiest thing is to increase the starting material. Can you do that? Which cell are you using? In my experience, loading 30µg of protein it´s enough to have a good WB. Also try to resuspend you pellet directly in lysis buffer as Tris-HCl SDS or RIPA 

Rossella

increase the amount of starting conditioning media ( always in ratio with the number of cells)  so you have more exosomes

when I start to work with a new cell line, I recover exosomes from 1, 2, 4 plates and then I resuspend the exosome pellet directly in Laemmli buffer and I run my Western blot. Afterwards, I know what is my minimal condition to get a good signal in WB without doing any quantification. I hope it helps

I used, 40ul of Lysis buffer directly after ultracentrifugation of 18 ml conditioned media and extracted proteins. The concentration of protein is higher in Lysis buffer than using RIPA buffer. Then, I used 10ug of protein to run a Western blot. It works good.

You mention you have collected exosomes from conditioning media (CM), the quantity of exosomes depend on your cell lines you use and also the volume of your CM, plus the method of isolation, so before you start any downstream analysis, you should consider these factors, for RNA extractions and qPCR is fine.

As some people have suggested, increasing the amount of starting material might be the best option. I used to collect the microvesicles/exosomes of ~8 T175 flasks (120-150ml) and resuspend the pellet after ultracentrifugation in ~100ul of RIPA buffer + protease inhibitors. Bear in mind that cell lines differ a lot in terms of production of extracellular vesicles.

Are you sure that you are working with exosomes and not with micro vesicles? If not just name them extracellular vesicles (EVs). However, just use an anti tetraspanin antibody (e.g. CD63) which usually can be found on most EVs for immunoprecipitation. Thus you do not have any problems with the volume. Good luck. 

I concur with the previous answers. I usually use 5 T175 flasks (100 ml media) as a starting point. I tested several cell lines and could observe huge differences in yield. It also seems to me that cell density is quite important. Too confluent cells secreted less EVs in my experiments. I would also suggest resuspending in RIPA, especially because EVs do not resuspend well in PBS. I then usually use 30-50 µg of protein per lane.

Hi,

Paying attention to all steps in this workflow is important in order to get good signal in WB in the end. I would therefor suggest the following:

1. Optimization of cell culture conditions.

I am adding our in house system as an example. Cell culture and exosome production

culture adherent cells to confluency (RPMI 1640 w/ 10% FCS, 1 mM Sodium-pyruvat in cell culture bottles (225 cm2) in 37C w/ 5% CO2.

remove medium form confluent cells and add 50 mL of fresh medium.

After 3 days the cell-conditioned medium is removed and two centrifugation steps are performed on the pre conditioned medium prior to pre-enrichment (300xg for 10 minutes at 2-8oC and 2000xg for 30 minutes 2-8oC).

2. Test for exosome release using magnetic beads (Dynabeads)

depending on the cell type and their exosome production efficiency it is possible to pull out exosomes from the solution directly without any pre-enrichment step and perform e.g. flow analysis. This is very useful in order to check the status of the cells exosome release efficiency and find the correct time for exosome harvest. I would use Dynabeads CD9, Dynabeads, CD63 or Dynabeads CD81 (hopefully your exosomes do contain at least one of these common exosomal markers (could stain the host cell first for all these markers and do flow analysis – at least it will give you an indication).

A flow protocol may look like this:

Capture for flow

The protocol is based on isolation using 200.000 magnetic beads but it can be scaled up by increasing volumes and reagents proportionally. 

Preparation of pre-enriched exosome sample

Use cell culture directly (pre-enriched exosomes can also be used to test the pre-enriched exosome preparation).

Add Isolation Buffer to 100 µL final volume per 20 µL magnetic beads. 

Day 1.

Resuspend the magnetic beads by mixing for >10 minutes or vortexing for 30 s.

Transfer 20 µL magnetic beads into an appropriate tube.

Wash the magnetic beads by adding 200 µL of Isolation Buffer and mix well.

Place the tube on the magnet for 1 min and discard the supernatant.

Remove the tube from the magnet, and add pre-enriched exosome solution titrated with Isolation Buffer (100 µL final volume) to the magnetic beads. Mix well.

Incubate the tube ON (18-22 h) at 2-8°C with mixing.

Day 2.

Centrifuge the tube for 3-5 seconds to collect the sample at the bottom of the tube.

Wash the bead-bound exosomes by adding 300 µL of Isolation Buffer. Mix gently by pipetting.

Place the tube on the magnet for 1 min and discard the supernatant.

Remove the tube from the magnet and add 400 µL of Isolation Buffer. Mix gently by pipetting.

Place the tube on the magnet for 1 minute and discard the supernatant.

Resuspend the bead bound exosomes in 300 µL Isolation Buffer.

The bead bound exosomes are now ready to be stained for flow cyt.

Staining for flow cytometry

The staining antibody should be titrated for optimal signal to noise ratio, starting with the manufacturer`s recommended concentration for staining 1 x 106 cells.

Transfer desired staining antibodies to a flow tube.

Add 100 µL bead-bound exosomes to the tube. Mix gently by pipetting.

Incubate for 45-60 minutes at RT protected from light on a sample shaker (approximately 1000 rpm).

Wash the bead-bound exosomes by adding 300 µL of Isolation Buffer. Mix gently by pipetting (do not vortex).

Place the tube on the magnet for 1 minute and discard the supernatant.

Repeat the washing steps once and resuspend in the desired volume of Isolation Buffer for flow cytometry analysis

3. Direct approach or a pre-enrichment strategy.

The flow signal you will get might vary from very low/nothing or up to signals that are actually sufficient for further detailed analysis. Depending on this signal you may decide to continue to do direct isolation with the Dynabeads as they are compatible with many different downstream applications such as western, electron microscopy, MS, seq, PCR etc. If the signal you get from the pre-testing by e.g. flow is very low you may consider doing a pre-enrichment step (UC or precipitation).

A precipitation protocol can look like this:

• Harvest media.
• Centrifuge at 350xg for 10 minutes.
• Transfer the supernatant to a new tube.
• Centrifuge supernatant at 2000xg in 30 min at 2-8C.
• Mix 1 part cell-free culture media to 0.5 volumes of the Total Exosome
• Isolation (from cell culture media) reagent.
• Mix well by vortexing, or pipetting up and down until there is a homogenous solution.
• Incubate samples at 2-8C ON.
• Centrifuge the samples at 2-8C at 10.000xg for 1 hour (fixed angle rotor).’
• Discard supernatant. Exosomes are in the pellet at the bottom of the tube for swing-out rotors or at the tube-wall near the bottom for fixed angle rotor.
• To remove liquid and leave the tube upside down on a filter paper for 3-4 min
• Resuspend the pellet in a given volume in 1xPBS.
• Exosomes are ready for downstream analysis or further purification through affinity methods or aliquotation and freezing in -80°C 

4. Western blotting from pre-enriched exosomes

As the amount of protein can be low I try to add all starting material in each well to get a proper signal. Currently we are using gels which have the capacity to add 40 µL of sample (Bolt system). The suggestion below is scaled according to this well size.

A WB protocol might look like this:

• 24µl pre-enriched exosome sample
• 6µl 5xRIPA with prot inhib
• 10s sonication

→ incubate 15 min at 4°C

Exosome boiling

• 30µl of lysed exosomes
• 10µl 4xLDS  

→ incubate 10 min at 70°C

Apply all in one well

5. Western Blotting – subpopulations of exosomes isolated with magnetic beads

If initial flow signal is fine it is possible to isolate directly from cell culture media

If initial flow signal is very low pre-enrichment should be considered.

If one wants to obtain ultra-pure exosomes – Dynabeads decorated with prim Abs will allow to recover very clean population of exosomes, following initial purification e.g with Total Exosome Isolation (from cell media) or ultracentrifugation – or directly from culture media. These subpopulations of exosomes can be analysed by electron microscopy, flow cytomtery, WB and RNA contect etc.

To maximize the WB signal in cases with reduced amount of exosomes oe may consider increasing the amount of magnetic beads used for isolation. This will provide a larger surface for exosomes to dock onto in addition to improve the binding kinetics. Increasing the number of beads will give an increasing signal on WB. We have also been able to pull out exosomes directly from cell culture media without any pre-enrichment after optimizing the cell culture growth conditions and exosome harvest conditions followed by WB.

A protocol for exosomes isolated with magnetic beads may look like this (20 µL beads + 100 µL sample volume):

Lysis of exosomes on Dynabeads & Boiling of exosomes after bead isolation

• All Dynabeads (when supernatant is removed)
• Add 6µl of 5xRIPA with prot inh (1.25 µL 25x)
• Add 24 µL dH2O
• 10 s of sonication
• Incubate for 15 min on ice
• Remove beads by applying magnet10 µL 4xLDS
• 10 min at 70°C
• Add 40 µL/well (4-12% bis tris gels)

Maximize Blotting conditions:

For blotting I would recommend wet transfer to PVDF membranes.

Maximize signal:

I would strongly recommend to use the most sensitive substrate – in-house we have had great success with the Super Signal West Dura as this gives high signal intensity and duration and is suitable for using in combination CCD cameras.

CD63 detection:

CD63 can in itself be a challenge to detect. The Abs targeting tetraspanins often require non-reducing conditions during sample prep for WB – so no mercapto during boiling of your sample. Furthermore, the CD63 clone is also important. According to key opinion leaders in this field the CD63 should give a smear on WB between 30-60 kDa. The clone preferred is TS63 which we have used as well during development – it can also detect different smear patterns depending on the exosome origin – indicating that the glycosylation pattern is different depending on origin. Interestingly, the CD63 expression also seems to vary a great deal between different sources.

CD81 and CD9 detection:

CD81 and CD9 detection might be worth including as well as they give single bands around 30 kDa (but not all exosomes are e.g. CD9 positive and also the expression level differs between different sources). 

We have Abs that have been validated for western blot detection of CD9, CD63 and CD81

kind regards

ketil 

Hi all,

Thank you so much for the great response!

I've tried to re-pellet my exosomes (resuspend in only 20ul of PBS) but I still did not get a high protein concentration. 

As many have suggested, I think its increasing the amount of starting material which is best and re-suspending directly in RIPA buffer. I will give this a go and see if I get a better yield. 

For those who asked I am using osteosarcoma cell lines MG63 and HOS. And its the first time I am working with exosomes. If anyone has worked with these yet, would be great to share some tips and suggestions :) 

Thanks again for the great response!

Kristina 

I usually use less amount to resuspend exosomes after ultracentrifugation.Also i counnt the number of exosomes for each samples and use that number to quantify my band intensity from western blot.

I hope this will be helpful.

I've had this problem too. Depending on the cell source you may have 2-3 orders of magnitude difference in secretion of exosomes per million cells. Doubling or tripling the starting material may be insufficient alone and you might have to try concentration of the exosomes in addition to increasing starting material.

Ketil's protocol - excellent in its entirety - suggested immunobeads which alone are a good answer to concentrating subset's of exosomes. You might also look into some of the proprietary kits such as Total Exosome Isolation reagent, ExoQuick or ExoSpin as post-ultracentrifuge concentration options, although - unlike immunobeads - those don't seem to effectively remove contaminating protein and may result in an overestimate of exosomal protein. Recently there have been a couple of papers - in the open access Journal of Extracellular Vesicles in particular - detailing simple size exclusion separations which seem to effectively concentrate exosomes into one or two fractions and to also 'clean' them of contaminating proteins. I suppose size exclusion gel columns could be scaled to process larger volumes of culture media if needed. Concentration of exosomes by ultrafiltration is also well described although I know less about that and have not used it.

One other thought is that a couple of studies have shown that using exosome-depleted serum versus serum free media can make a significant difference to recovered exosome number, although there doesn't seem to be agreement between publications regarding which is better; like most other things, that issue is likely cell-type and culture-condition specific!

Ben

Hello everyone,

Happy to say your suggestions worked. I started with around 5 T175 flasks, purified the exosomes by ultracentrifugation and resuspended the exosome pellet directly in RIPA buffer. I loaded 5ug/uL and got nice clean bands for CD9 and CD63. 

Kristina 

Wonderful, just please make sure to use very optimized media to grow your cells for exosome preparations, some media have bovine fetal serum, and the reality people think they got a good quantity of exosomes, but eventually they isolated from serum and not form cell culture media, good luck!!

Dakir

Hello everyone,

As everyone have mentioned already mentioned about increasing the starting material in order to obtain high yield.

I have a small question about what do you consider as high yield ?

In terms of "protein concentration", how much exosome protein equivalent concentration do you expect from 240ml of conditioned medium ?

Thanks

Would you please provide me the antibody information? I also have samilar problem, I was thinking maybe it's because of the antibodies?

Thanks a lot

shunfu