We are performing in vitro studies on graphene-based nanoparticles using CD34+ HSC's. We wish to perform TEM to confirm cellular uptake of the NP's.
My colleague is concerned that extracellular NP's will affect the TEM- for example: extracellular NP's out of plane with a cell might look as though they were within the cell. I have performed similar studies with adherent cells; a simple PBS wash was enough to remove extracellular NP's. However, since the HSC's are nonadherent, we cannot use this protocol.
Does anyone know of a good way to separate the extracellular NP's and cells?
You can spin down (centrifuge) the cell sample which leaves free NPs in solution and create your thin sections from the collected cells. Cell membrane adherent NPs can follow the cells. With TEM you have sufficient resolution to distinguish NPs in the cell lumen from NPs attached to the membrane, although with carbon based NPs your contrast will be very low. I will be interested in how well you manage to resolve them. We always use several techniques to make sure that we do not count artefacts in the TEM.
Eric, thanks for your answer. We considered spinning down the sample, but wouldn't the NP's also collect on the bottom of the tube with the cell pellet? We also considered using a filter in the centrifuge, but we do not have any with sufficiently small pore size to trap the HSC's.
Typically not, as the method separates by size and the size difference is large. But if you think you have that problem, yes, then you can improve by using a filter tube or density gradient. There are plenty of resources from e.g. commercial suppliers of equipment and such that could aid you in that choice if you need it / like it.
Dear Sirotkin,
I thin you should contact Dr madhuri Sharon, email: [email protected] She works in this area and she may be able to give you some tip. centrifugation may or may not seprate NP with cell. in fact it may do some damage.
Sharon
Washing the media to remove extra cellular NPs might be a good option, but be careful, as the washout may cause the NPs to be released from the cells and hence total internal NP concentration may be lowered in some cell types. Centrifugation will not cause the NPs to be pelleted along with the cells, as you need high speed to bring the NPs to the bottom. Try to centrifuge the cells at around 2000 RPM for 2-3 mins, or 1000 RPM for 4-5 mins.
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