I apply the vacuum for 20 sec but I don't know whether -90 KPa...

Hi Chuan Shen,

Vacuum infiltration will very depending on the strength of you vacuum pump. I would say as long as your plants are being uniformly infiltrated and aren't leaking chloroplast into your wash fluids (as indicated by a green color) it's probably okay.

It shouldn't have an effect on your results whether you use 2*C or 4*C. You merely want to keep the EVs stable in a cool temperature while centrifuging.

I use MES (pH 6) when initially isolating EVs or handling them in general. This buffer is isotonic and was designed to match the pH of the apoplast, which is slightly acidic. I use Tris-HCl (pH 7.5) when staining with DiOC6, just in case an acidic pH affects DiOC6 fluorescence. The EVs are stable in either buffer.

I think your problem is probably in your technique and the equipment you're using during the ultracentrifuge steps. I use a tabletop ultracentrifuge and 3.5 ml thick wall, polycarbonate tubes with a fixed-angle rotor. I always make sure the tube contains its maximum volume. This prevents cracking of the tubes and washes away impurities. I also decant the supernatant. If you're using a swinging bucket rotor, this creates a loose pellet that can be easily lost. The shape of the tube can also affect the quality of your pellet, as well as pipetting out too much of the supernatant. Unfortunately, the pellet is invisible except when using very high amounts of apoplastic wash, so you'll have to guess where it is.

I would recommend going over you technique. Try looking at Thery et al. (2006). It has lots of helpful tips on technique for isolating EVs: Théry, Clotilde, et al. "Isolation and characterization of exosomes from cell culture supernatants and biological fluids." Current protocols in cell biology 30.1 (2006): 3-22.

If you're losing the majority of your EVs through your technique, you might also try using higher amounts of wash fluid.

Best of luck,
Brian