Posts Tagged: methods

Methodological Guidelines to Study Extracellular Vesicles

Methodological Guidelines to Study Extracellular Vesicles

Frank A.W. Coumans, Alain R. Brisson, Edit I. Buzas, Françoise Dignat-George, Esther E.E. Drees, Samir El-Andaloussi, Costanza Emanueli, Aleksandra Gasecka, An Hendrix, Andrew F. Hill, Romaric Lacroix, Yi Lee, Ton G. van Leeuwen, Nigel Mackman, Imre Mäger, John P. Nolan, Edwin van der Pol, D. Michiel Pegtel, Susmita Sahoo, Pia R.M. Siljander, Guus Sturk, Olivier de Wever, Rienk Nieuwland

Working principle of common methods to isolate extracellular vesicles (EVs). Separation is based on size, density, and immunophenotype. Straight brackets: isolated EVs; yellow: soluble components; and blue: buffer. A, In differential centrifugation, separation is based on size, and large EVs (gray) collect earlier at the bottom of the tube and at lower g forces than small EVs (green). The soluble components are not affected by centrifugation, but non-EV particles such as lipoproteins and protein aggregates may copellet with EVs. B, In density gradient centrifugation, separation is based on density, and EVs will travel to their equilibrium density. Non-EV particles such as lipoproteins may coelute with EVs because of similar density or interaction. The soluble components with a high density relative to the gradient will collect at the bottom of the tube. C, Size exclusion chromatography uses a porous matrix (dotted circles) that separates on size. Soluble components and particles smaller than the size cutoff enter the porous matrix temporarily, whereas EVs and particles larger than the size cutoff do not enter the porous matrix. As a result, EVs and particles larger than the size cutoff elute before the soluble components and particles smaller than the size cut-off. D, In ultrafiltration, soluble proteins and particles smaller than the size cutoff (≈105 kDa) are pushed through the filter, and the EVs are collected at the filter. E, In immunocapture assays, EVs are captured based on their immunophenotype. EVs are captured using an monoclonal antibody (mAb) directed against an antigen exposed on the targeted (green) EVs only. F, In precipitation, addition of a precipitating agent induces clumping of EVs, non-EV particles, and soluble proteins. The clumps will sediment, and sedimentation can be accelerated by centrifugation. [Powerpoint File]

Untangling Autophagy Measurements: All Fluxed Up

Untangling Autophagy Measurements: All Fluxed Up

Roberta A. Gottlieb, Allen M. Andres, Jon Sin, David P.J. Taylor

Autophagic puncta in hearts of mCherry-light chain 3 mice on chow or high-fat diet (HFD). A, Representative heart sections of mice from the Mentzer study after 15 weeks of chow or Teklad D12492 HFD. Cardiac sections (5–6 μm) were made using a cryostat, mounted on glass slides, and nuclei stained with Hoechst 33342. Images were acquired with a Nikon TE300 fluorescence microscope equipped with a ×60 Plan Apo objective with excitation/emission wavelengths of 560/630 nm (unpublished images generously provided by Dr Bruce Ito and Dr Robert Mentzer). B, Representative heart sections of mice from the Abel study after 12 weeks of chow or Western diet. Images were taken with Zeiss LSM 510 confocal microscope using ×63 immersion oil objective with excitation/emission wavelengths of 543/613 nm (unpublished images generously provided by Dr Bharat Jaishy and Dr E. Dale Abel). [Powerpoint File]