MVs were isolated from differentiating chondrocytes by differential centrifugation as described earlier.(9 (link)) A drop (5 µL) of each MV solution in Tris-buffered-saline was spotted on freshly cleaved mica substrates (Ted Pella, Redding, CA) and allowed to stand for 5 min. Next, 5 µL of glutaraldehyde solution (8% in H2O, Sigma-Aldrich, St. Louis, MO) was dropped onto the samples. The substrates were stored inside a desiccators at room temperature for 24 h. AFM images of dried samples were recorded in air by means of an 5500 atomic force microscope (Agilent Technologies, Santa Clara, CA) equipped with an open-loop probe working in non-contact (AAC) mode. Silicon-nitride cantilevers having a nominal resonance frequency of ~190 kHz (NanosensorsTM, Neuchatel, Switzerland) were used. Tridimensional AFM images were generated by PicoView software (Agilent Technologies). AFM images were used to gather information about the morphology, height, volume and number of MVs in each sample. AFM phase images were also recorded on samples prepared without the use of glutaraldehyde.
Protocol detail
Atomic Force Microscopy of Chondrocyte-Derived Extracellular Vesicles
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Atomic force microscope
Centrifugation
Chondrocytes
Glutaraldehyde
Mica
Resonance
Saline solution
Silicon nitride
Corresponding Organization : Sanford Burnham Prebys Medical Discovery Institute
Other organizations : University of Rome Tor Vergata, La Jolla Bioengineering Institute, University of California, San Diego, Karolinska Institutet, Inserm, Le Laboratoire d'Ingénierie Ostéo-Articulaire et Dentaire, Roslin Institute, University of Edinburgh
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Variable analysis
independent variables
- Isolation of MVs from differentiating chondrocytes by differential centrifugation
- Morphology of MVs
- Height of MVs
- Volume of MVs
- Number of MVs in each sample
- Tris-buffered-saline used as the solution for MV samples
- Freshly cleaved mica substrates used as the surface for MV samples
- Glutaraldehyde solution (8% in H2O) used for fixation of MV samples
- Samples stored inside a desiccator at room temperature for 24 h
- AFM images recorded in air using an Agilent 5500 AFM in non-contact (AAC) mode
- Silicon-nitride cantilevers with a nominal resonance frequency of ~190 kHz used for imaging
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
- AFM phase images recorded on samples prepared without the use of glutaraldehyde
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