<p class='jove_title'><strong>1. Block Preparation of Exosomes</strong></p>
<ol>
	<li>Pellet the exosomes from the culture supernatant of HCT116 cells by centrifugation at 100,000 x <em>g</em> for 1.5 h. Remove the culture supernatant and carefully fix the purified exosome pellet with 1 mL of 2.5% glutaraldehyde in 0.1 M sodium cacodylate solution (pH 7.0) for 1 h at 4 &#176;C. For 0.1 M sodium cacodylate, dissolve 4.28 g cacodylic acid in 160 mL of distilled water (DW). Adjust pH to 7.4 with 0.1 M HCl, then make up to 200 mL with distilled water.</li>
	<li>Remove the fixative and rinse the pellets with 1 mL of 0.1 M sodium cacodylate buffer at room temperature. Repeat three times, with each change lasting 10 min.</li>
	<li>Post-fix the sample with 1 mL of 2% osmium tetroxide for 1 h at 4 &#176;C.</li>
	<li>Remove the fixative and rinse three times with 0.1 M sodium cacodylate buffer every 10 min.</li>
	<li>Incubate for 10 min with a graded acetone series (50%, 60%, 70%, 80%, 90%, 95%, 100%, respectively) on the shaker.</li>
	<li>Remove the acetone and incubate the solution of 3:1 acetone:low viscosity embedding mixture for 30 min. The exosome pellet is in the tube.</li>
	<li>Remove the medium and add 1:1 acetone:low viscosity embedding mixture medium, then incubate for 30 min.</li>
	<li>Remove the medium and add 1:3 acetone:low viscosity embedding mixture medium, then incubate for 30 min.</li>
	<li>Remove the medium and add 100% low viscosity embedding mixture and incubate overnight at room temperature.</li>
	<li>Embed the sample in pure low viscosity embedding mixture using the embedding mold and bake for 24 h at 65 &#176;C.</li>
</ol>

<table><tbody><tr><td>Glutaraldehyde&amp;nbsp;&amp;nbsp;</td><td>EMS</td><td>16200</td><td></td></tr><tr><td>Sodium cacodylate&amp;nbsp;&amp;nbsp;</td><td>EMS</td><td>12300</td><td></td></tr><tr><td>Osmium tetroxide 1 g crystal&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>EMS</td><td>19100</td><td>Use only in fume hood</td></tr><tr><td>Acetone</td><td>JUNSEI&amp;nbsp;</td><td>1B2031</td><td></td></tr></tbody></table>

exosome block preparation: a protocol to preserve exosomes from cultured human colon cancer cells

Exosome Block Preparation: A Protocol to Preserve Exosomes From Cultured Human Colon Cancer Cells

<p class='jove_content'>Source: Jung, M. K. <em>et al</em>. <a href='https://www.jove.com/video/56482' target='_blank'>Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy.</a> <em>J. Vis. Exp.</em> (2018)</p>
<p class='jove_content'>This video describes the technique of preparing exosome blocks from cultured gastrointestinal tumor cells. These exosome blocks can further be sliced, stained, and imaged to determine the position of specific proteins in the lipid bilayer vesicle.</p>

Source: Jung, M. K. et al. Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy. J. Vis. Exp. (2018)
This video describes the ...

<p>Exosomes are the extracellular vesicular structures that are secreted by cultured cells into the supernatant. To prepare an exosome block, begin by taking an exosome-containing culture supernatant in a tube. Centrifuge the tube at high speed to pelletize the intact exosomes. Discard the remaining supernatant.</p>
<p>Treat the exosome pellet with glutaraldehyde present in the fixing buffer. Incubate the mix on ice for the desired time to preserve the morphology of exosomes. Glutaraldehyde infiltrates the exosomes and crosslinks the proteins. Fixing buffer maintains exosomes at neutral pH for the downstream fixation process.</p>
<p>Discard the glutaraldehyde-containing buffer and add osmium tetroxide solution - a secondary fixative that permeabilizes the membrane bilayer and fixes the lipids present in exosomes. Remove the osmium tetroxide solution and treat the exosomes with a series of increasing concentrations of acetone. This process dehydrates the exosomes.</p>
<p>Replace absolute acetone with the embedding medium-acetone mixture. Gradually, increase the ratio of embedding medium in this mixture.</p>
<p>Pour the embedding mixture containing exosomes into desired molds. Oven-dry the molds at a high temperature. This will lead to polymerization of the embedding medium around the exosomes, giving rise to blocks.</p>

<p>To fix the purified exosome pellet, add 1 milliliter of 2.5% glutaraldehyde in a 0.1 M sodium cacodylate solution at pH 7.0. Incubate the lipid bilayer vesicles for 1 hour at 4 degrees Celsius. Next, remove the fixative and rinse the pellets with 1 milliliter of a 0.1 M sodium cacodylate buffer solution at room temperature for 10 minutes.</p>
<p>Then, post-fix the samples with 1 milliliter of 2% osmium tetroxide for 1 hour at 4 degrees Celsius. Remove the fixative and rinse the exosome pellet three times with the 0.1 M sodium cacodylate buffer, changing the buffer every 10 minutes. Then, dehydrate the sample by shaking it for 10 minutes each in a series of graded acetone concentrations.</p>
<p>Next, mix up a solution of three parts acetone and one part of low viscosity embedding mixture. Replace the acetone with this mixture and incubate the sample for 30 minutes. Continue to increase the ratio of the low viscosity embedding medium, incubating for 30 minutes with each step.</p>
<p>Finally, incubate the exosome pellet in 100% of the low viscosity embedding mixture overnight at room temperature. The following day, embed the sample in pure low viscosity embedding mixture using an embedding mold and bake it for 24 hours at 65 degrees Celsius.</p>

exosome-block-preparation-protocol-to-preserve-exosomes-from-cultured

Research

JoVE Encyclopedia of Experiments

Cancer Research

Gastrointestinal Cancer

Assays and techniques

3.7K Views. Exosomes are the extracellular vesicular structures that are secreted by cultured cells into the supernatant. To prepare an exosome block, begin by taking an exosome-containing culture supernatant in a tube. Centrifuge the tube at high speed to pelletize the intact exosomes. Discard the remaining supernatant. Treat the exosome pellet with glutaraldehyde present in the fixing buffer. Incubate the mix on ice for the desired time to preserve the morphology of exosomes. Glutaraldehyde infi...

Abstract

Protocol

Disclosures

<table><tbody><tr><td>Glutaraldehyde&nbsp;&nbsp;</td><td>EMS</td><td>16200</td><td></td></tr><tr><td>Sodium cacodylate&nbsp;&nbsp;</td><td>EMS</td><td>12300</td><td></td></tr><tr><td>Osmium tetroxide 1 g crystal&nbsp;&nbsp;&nbsp;</td><td>EMS</td><td>19100</td><td>Use only in fume hood</td></tr><tr><td>Acetone</td><td>JUNSEI&nbsp;</td><td>1B2031</td><td></td></tr></tbody></table>

Exosome Block Preparation: A Protocol to Preserve Exosomes From Cultured Human Colon Cancer Cells

In This Article

Abstract

Protocol

Disclosures

Materials

Tags

Name	Company	Catalog Number	Comments
Glutaraldehyde	EMS	16200
Sodium cacodylate	EMS	12300
Osmium tetroxide 1 g crystal	EMS	19100	Use only in fume hood
Acetone	JUNSEI	1B2031