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Non-Hazardous Dehydrated Culture Media SDS

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  • R452952
  • CM1012(Discontinued)
  • CM1076
  • CM1079
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  • CM0785
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  • CM0911

Oxoid non-hazardous Dehydrated Culture media [AUS] SDS

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Oxoid non-hazardous Dehydrated Culture media [NZ] SDS

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Oxoid Culture Media product[JA] Product Literature

How can I get a good TEM picture of my exosomes? Product FAQ

Answer

We have standardized dissolving the pellet of cell culture exosomes after isolation with Total Exosome Isolation reagent as follows:

Beckman J2-21M/E centrifuge with JA20 rotor
Nalgene centrifuge tubes for Beckman centrifuge
PBS, 0.22 µm filtered
- Start with 20-25 mL of cell culture supernatant from overnight isolation (using Total Exosome Isolation reagent).
- Centrifuge at 10,000 x g = 9200 rpm for 1 hour (set temperature at 10 degrees C, as lower setting may result in temperature as low as 0-2 degrees C during centrifugation).
- Remove supernatant by suction.
- Leave tubes upside-down on absorbant paper for 10 min at RT to remove residual buffer.
- Add buffer to cover the pellet when the tubes are placed at an angle to the bench (500 µl PBS/sample tube), leave for 30 min at RT.
- Carefully resuspend by pipetting and pool sample from each centrifuge tube.
- Collect residual volumes from each sample tube by centrifugation for 8 min at 350 x g.
- Aliquot and freeze at -80 degrees C.

Exosomes can be processed for electron microscopy for ultrastructural analysis both prior to Dynabeads magentic beads isolation and after isolation. Prior to isolation the exosomes pool can be immunolabeled and processed for negative stain prior to ultrastructural analysis. Such a protocol could look like this:

- Load exosomes undiluted at RT for 15 min.
- Block with 0.5% BSA for 10 min.
- Label with primary antibody for 30 min.
- Wash 5X with PBS for 10 min total.
- R&M (1:100) for 30 min.
- Wash 5X with PBS for 10 min total.
- Prot A Au 10nm for 15 min.
- Wash 5X with PBS for 10 min total.
- Wash 5X with water for 10 min total.
- Embed in 0.3% uranyl acetate in methyl cellulose.
- Conduct TEM analysis.

Subpopulations of exosomes prepared using the Total Exosome Isolation kit or ultracentrifugation can also be processed for ultrathin sectioning and electron microscopy. After isolation and washing of the exosomes on the surface of the Dynabeads magnetic beads, they can be processed using the traditional TEM protocol described by Pedersen et. al. in J Virol (1999) 73:2016–2026. In brief, for conventional Epon embedding and sectioning, Dynabeads magnetic beads with exosomes can be fixed for 1 hr in 1% glutaraldehyde in 200 mM cacodylate buffer (pH 7.4), washed repeatedly in aqua destillata, and incubated for 1 hr in cacodylate buffer containing 1% OsO4 and 1.5% K3Fe(CN)6. Following two subsequent 30-min incubations in 1% tannic acid and 1.5% magnesium uranyl acetate, the samples are dehydrated by using ethanol and embedded in Epon. Ultrathin sections can be stained with lead citrate.

Answer Id:: E7402

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