I'm in the process of isolating rat pan T lymphocytes from rat spleens, and intend to use Miltenyi's rat pan T lymhocyte microbeads for this, as these are one of the only on the market that are rat specific. However, we only have an Invitrogen Dynal magnet in the lab, and I was hoping that this might work, despite the significant size difference in the beads (Miltenyi's microbeads are 50 nm, whereas Invitrogen's Dynabeads are 1 micron). Has anybody here done this before?
For Positive selection with Invitrogen Dynabeads, the kit provides you a cell release buffer or detach bead solution. However, positive selection with MACS microbeads works when you load cells onto a MACS LS column and you need a MACS magnet to hold the column so that when plunged with buffer the positively selected cells are released. I am not sure if cells stained with MACS microbeads can be released using just Dynal magnet as you need to release the cells with a dissociation buffer which does not come with MACS microbead kits.
I have never done it but I guess that if the beads are magnetic, they should get retained in the magnet regardles of their size. For releasing the cells I dont think you should have any problem. I think that you could use PBS or an specifuc media for you cells.
I have tried it in the past with other cell isolation nanoparticles and did not get good results. A Dynabead magnet did not work with nanoparticles. I needed the Miltenyi columns, specifically. to get good separation with magnetic nanoparticles.
The best results are generally obtained with the best matched tools but ... if you can not do differently (?). First check whether the Miltenyi nanoparticles alone are attracted by the Dynal magnet. This may require more time than with Dynabeads (e.g. 10 min versus 1 min) but it may (or may not?) finally work. At least it is quick and easy to check. Several years ago, I had been usefully surprised to easily catch endothelial cells recognized in whole blood by CD146 MAb-coated so-called "Ferrofluids" (magnetic beads of ~200 nm) using a Dynal magnet, although I would not have bet for it before trying.
Another trick (theory-based suggestion, never tested yet). Add uncoated/plain magnetic Dynabeads in your cell suspension, in addition to the Miltenyi nanobeads. The microbeads may well bring the magnetic fielld closer to the nanobeads and do the same job as the metallic sieve included in the Miltenyi colums that drive the magnetic field closer to the nanobeads. Keep us aware of your trials.
Thanks everyone! I've managed to borrow a MACS magnet and column from some friendly neighbours.
Dear Jennifer
I have never done it but i read some protocol and article about size of iron particle the size of particle have relation with affinity of magnet and iron particle
if the size is smaller affinity get more and this effect on your result
best wishes
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