Manual / Product Insert

User Guide: MagMAX Cell-Free DNA Isolation Kit

Version: E.0
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Manual / Product Insert

Long Protocol: Cancer Cell Isolation Kit

Version: JAN 2017
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Manual / Product Insert

User Guide: MagMAX Cell-Free Total Nucleic Acid Isolation Kit

Version: B.0
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Manual / Product Insert

User Guide: Pierce Primary Neuron Isolation Kit

Version: MAN0016221 Rev. A.0 (03Oct2016)
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Manual / Product Insert

User Guide: Pierce Cell Surface Protein Isolation Kit

Version: Jan. 2015
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Manual / Product Insert

User Guide: Pierce Primary Cardiomyocyte Isolation Kit

Version: MAN0016222 Rev. A.0 (03Oct2016)
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Manual / Product Insert

User Guide: Primary Mouse Embryonic Fibroblast Isolation Kit

Version: MAN0016041 Rev. A.0 (19Jul2016)
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Product FAQ

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

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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Product FAQ

Can you provide an overview of epithelial cell enrichment using the CELLection Epithelial Enrich kit?

Answer

CELLection Epithelial Enrich reagent is bound to the beads via a cleavable DNA linker. This provides you with the option to release the isolated cells from the beads using the supplied release buffer, which returns cells with no beads attached for further cell culture or functional assays. CELLection Epithelial Enrich reagent is recommended for processing a total of 2 x 10E9 cells. The CELLection Epithelial Enrich beads are coated with a mouse anti-BerEP4 monoclonal antibody to the human epithelial cell adhesion molecule (EpCAM).

By making a single-cell suspension of the tissue, it should be possible to isolate the cells using CELLection Epithelial Enrich reagent (for cellular applications) or Dynabeads Epithelial Enrich reagent (for molecular applications). The difference between the CELLection Epithelial Enrich product and the Dynabeads Epithelial Enrich product is that the primary antibody on the CELLection product is coupled to the beads via a DNA linker, providing this bead with a release mechanism via the DNase containing release buffer supplied with the kit. This product is intended for isolation of cells that need to be bead free for downstream studies, while the Dynabeads Epithelial Enrich product is intended for molecular applications (e.g. DNA or mRNA isolation). However, since the antibody coated onto these beads recognizes EpCAM, this epitope needs to be expressed on the cells.

The CELLection Epithelial Enrich product is intended for enrichment of tumor cells rather than isolation of pure cells. This is because the number of target cells can often be as low as 1 tumor cell in one million cells, making it very difficult to get rid of all the contaminating cells even after rigorous washing. This is why we also recommend that molecular analysis or visual morphological verification is necessary to verify that the isolated cells are indeed tumor cells.

Answer Id: E6117

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Manual / Product Insert

PichiaPink Expression System

Version: MAN0000717 A.00 (22 Jan 14)

Product Literature

Surface Activated Dynabeads

Manual / Product Insert

Dynabeads Rat anti-Mouse IgM

Version: 3.00
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Product Literature

Journal: BioProbes 80 Journal Of Cell Biology Applications

Manual / Product Insert

Instruction Manual: mirVana™ Probe & Marker Kit

Version: 1554M Revision C 06.2012
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