Wet lab. It was horrible. 6 years of dishwashing and monotony. I write software now and am much happier. The numbers were very difficult to reproduce and sensi

Wet lab. It was horrible. 6 years of dishwashing and monotony. I write software now and am much happier. The numbers were very difficult to reproduce and sensitive to tiny variations in experimental conditions, to the point of mystery. My MSc took me 6 years rather than 2, and that’s with a >90% scope cut. I can say that there was a “comparable” proportion of freeMitos to mitoMPs in the samples I was studying, which were induced from 786-O and 786-O/VHL cell cultures using a variety of agonists (atypically low concentrations of A23187, compared to literature, and serum-starved cell culture medium, seemed to work best; usual concentrations of A23187 seemed very violent on the cells, which is relevant if you’re trying to ascertain anything about physiologic conditions). Shockingly, a very sizeable proportion (10-50% of EVs above a certain detection threshold, probably near 100-150nm) of total EVs were either mitoMPs or freeMitos. I did not study mitoribosomes. I was trying to optimize conditions for the generation of EVs (literature is lacking here), enrichment of samples, and flow cytometric techniques for measuring EV subpopulations. Mitochondria make everything much more complicated and even gum up the tubes, requiring special care. Experiments that measure EVs without controlling for the presence of mitochondria are IMO inherently suspect. Their prevalence will depend on sample types, but from what I’ve seen, I believe the default assumption should be that they are present in relevant amounts. I’ve observed similar subpopulations in other cell lines, but with fewer controls and validations (I was focused on 786-O cells). There is a really great paper published by L Boudreau in the journal Blood with better results than anything I ever achieved, it’s very interesting stuff: https://pubmed.ncbi.nlm.nih.gov/25082876/ https://pubmed.ncbi.nlm.nih.gov/25082876/