Troubleshooting is going to be tough becuase there are no protocol details. I'm assuming that you did everything as the paper suggested? Because usually the first thing I always check with any flow analysis, is to ask if you are setitng your flow parameters correctly.

The easiest way to answer your question is to contact the authors directly. They know their protocol the best, and they can probably give you more details about the protocol than what was published.

I agree with Shen-An about checking flow parameters. Let's assume your exosome purification is not the problem.

First, I would make sure your exosomes survived the flow staining process. Prepare them exactly as you would for flow, but then view them with Nanosight. Here if there are problems, centrifugation and pipetting steps might need modification.

Once you're sure your Flow prep has not lost or damaged your exosomes, then optimize your flow cytometer settings. I have not specifically worked with exosomes, but based on their biology I would presume that several flow cytometer adjustments might be needed, especially if the flow cytometer is optimized for cells.

Find yourself a flow cytometer instrument expert to help. Variables such as flow rate, microfluidic droplet size, scatter size gate and laser voltages might need some adjusting!

Hi all,

Thanks for your prompt response. I have validated my exosomes by NanoSight and they are present and at the right size. If I were to prep it the way I would for flow, then the aldehyde sulfate beads which I use for flow would interfer with the readings. But I do think my flow prep might result in loss of exosomes. After I've incubated the beads to the exosome overnight, I centrifuge at 500 G for 5 mins. Perhaps I'm losing my exosomes in that process? I also block, wash twice, then stain and wash again. I have a feeling that my exosomes are lost in the washing and centrifugation steps. Is that a possibility?

I think it the next step is to ask the authors of the paper for their exact protocol.

So... I don't think your centrifuge speed is enough. For 4 um beads... you probably need somewhere around 3000gs. There is a link for reference. But I would just email the authors to get the details.

https://www.bangslabs.com/sites/default/files/imce/docs/TechNote%20203%20Web.pdf

^ agreed. Those centrifuge settings work for whole cells, but exosomes will require higher speeds/time. Especially since flow staining has many washes (even with whole cells you can lose some at each wash).

Use a vacuum aspirator when removing FACS staining buffer, instead of using a manual hand pipettor. The up and down motion when pipetting disturbs cell pellets, and exosomes might be even more prone to this.

One other tip: Sometimes a bucket centrifuge (as opposed to a benchtop microcentrifuge) helps minimize cell loss, because you get a nice pellet centered at the bottom of the tube, as opposed to on the side, where it's more vulnerable to aspiration.

Hi,

In terms of optimizing the conditions for flow and WB analysis we pay attention to the bead concentration. For flow we use as few Dynabeads™ as possible in order to have as many exosomes per bead as possible - to provide a strong flow signal (in setting we have of course not isolated the exosomes from the sample but rather captured enough to be analyze by flow - I like to think of it in a similar way as when you measure the pH of a solution). For WB we use as many Dynabeads™ as possible in order to provide a large total surface for exosome docking. In these cases I am not concerned about the number of exosomes/bead but rather to capture as many as possible (in this sense we have isolated the exosomes).This should give a good signal in WB. If you for some reason use the beads in this way and introduce them into flow, the signal will be very low.

I hope this will be of help

feel free to get in touch

ketil