Ev labeling methods and cautions learned from flow cytometric analysis of single evs by xiaomei yan
Notes
Notes on the WebEVTalk by Xiaomei Yan
Topics: extracellular vesicles, fluorescent labeling of extracellular vesicles, flow cytometer
- Welsh et al 2020: MIFlowCyt-EV: standardizing the report of extracellular vesicle flow cytometry data.
- Using cryo-TEM, 66% of EV's are below 100nm. 26% are between 100nm and 300nm. Only 8% are above 300nm (HCT15 cells). In urine, 77% are below 100nm.
- At 10^9 pcles/ml, there are 10^7 EVs/ml bigger than 300nm. This is enough for flow cytometry, but it misses the majority
- Philippe Poncelet (BioCytex), Flow cytometry for EV's is like an iceberg
- 25nm SiO2, light-producing power ~0.4 alexa fluorophor
- 60nm PS particle @532nm is 4X brighter than SiO2 particle.
- A SiO2 is 3.5 times brighter than an EV.
- nFCM can measure down to 40nm EV.
- Rayleigh scattering and fluorescence detection: detection of single SiO2 down to 25nm, gold down to 7nm
- Fluo limit, 3 Alexa Fluorophores
- Detection limit: 40nm EV, 10.000 particles/min
- Resolution similar to cryo-EM
- Side-scattering and two fluorescence
- Platelet-free-plasma, better percentage of EV
- Silicon NP to calibrate. Use the burst area and compare.
- Refractive index of EVs: 1.400, of Si: 1.461
- Calibration curve must correct for difference of refractive index
- Slide 16: a lot of particles are cramped at the lower limit.
- CryoEM takes 2 days
- Slide 17: Comparison of techniques. TEM took 8 hours, great resolution. nFCM, 2-3min, comparable to TEM. NTA 5-10min, impossible to resolve different particles.
- for EVs: NTA shows a peak at 105nm, while the true peak is at 55nm. CryoEM took 1-2 days
- Surface proteins of EV's. CD9, CD63, CD81. Most popular markers for EV's. ~Single-fluorophore detection.
- Quantification of protein expression on single-EV's.
- Purification is highly dependent on methods. Even depleting EV's from plasma gives some EV's while measuring (sometimes like the depletion didn't work at all)
- Concentrations of EV's vary wildly depending on method for purification.
https://www.youtube.com/watch?v=jLjyjPqXRO4
Backlinks
These are the other notes that link to this one.
Comment
Share your thoughts on this note. Comments are not public, they are
messages sent directly to my inbox.