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