Dear colleagues,
Has anyone of you ever experienced difficulties with the high background of the solvent-PBS (used to suspend the exosomes after isolation by ultracentrifugation) during NTA analysis by NanoSight NS300? The nanoparticles detected and measured in solvent-PBS (as a control of the measurement) by NTA are exosomes-sized (within the range of about 50-200 nm). The filtration by using 0.1 µm filters does not work at all. How to overcome this problem? How to prepare the PBS (or any other solvent?) for suspending the pellet of exosomes to avoid introduction other nanoparticles interfering with the NTA measurement ? I would be very grateful for any suggestions.
With kind regards,
Sylwia Katarzyna Król, PhD
Sylwia Katarzyna Król
One can sterilize the solution by filtering through a 0.1-μm filter unit. Filter sterilization removes all suspended particles with a size of more than 0.1 μm which includes most bacteria and their spores but not mycoplasma. Moreover, it does not inactivate enzymatic activities (e.g., DNases). Autoclaving inactivates most enzymes except some (e.g., RNases) and kills most microorganisms including mycoplasma.
https://www.laboratorynotes.com/preparation-of-10x-phosphate-buffered-saline-pbs-sambrook-method/
Dear Sylwia,
Your question is quite complicated in general and also poorly covered in either Nanosight/Malvern manuals or scientific papers. Good for you that you've started your NTA measurement with buffer blanks, because it's a very common problem that the data reported for EVs is in reality adventitious nanoparticles in buffer or media.
I researched this topic for long time in order to publish the paper, but unfortunately I've switched to other scientific topics. So, I'll try briefly share some key tips and tricks here.
First, you need a plentiful source of particle-free water. Typically, well-maintained MilliQ (18.2 MOhm*cm) is Ok. Try this water first, and do not continue until you're satisfied with water.
By saying "try" I mean introduce it into the instrument several times and check the amount of particles in a stopped flow. Less then 1 particle per frame in average (because sometimes there will be no particles, sometimes 1 or two) is Ok. If you're pumping the water through the instrument (with built-in pumps of NS500, or syringe pump for LM10, or manually with a syringe), there always will be some particles. This is unavoidable. So, once again, the criterion of water (buffer) purity is the absence (
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