I am really not an expert in this, but I would favour a negative selection strategy (i.e. removing all cells, except your target cells), since anything binding to a target cell, especially a lymphycyte can change a lot of things within milliseconds - there are many ways of "activation" of a cell - and there are many different receptors on your cells which can give some strange signals into the cell. But even any handling process can change the outcome of an experiment. I heard of an experiment (not involving T-cells, but mammalian cells: the student added some substance to the sample dishes, but wanted to gain time and did NOT treat the control dishes with PBS or include the control washing steps, which he did for the real samples; after weeks he learned that the activation he found was only due to the shaking of the cells and not the substance...)

Since changes can happen within milliseconds you should reflect this, if your outcome is questionable with the positive selection method, which may be OK depending on your experimental question, but I think any T-cell binding to anything gets strangely (non-specifically) activated. So maybe Dynabeads "untouched" or similar may work better for your approach.

I am really not an expert in this, but I would favour a negative selection strategy (i.e. removing all cells, except your target cells), since anything binding to a target cell, especially a lymphycyte can change a lot of things within milliseconds - there are many ways of "activation" of a cell - and there are many different receptors on your cells which can give some strange signals into the cell. But even any handling process can change the outcome of an experiment. I heard of an experiment (not involving T-cells, but mammalian cells: the student added some substance to the sample dishes, but wanted to gain time and did NOT treat the control dishes with PBS or include the control washing steps, which he did for the real samples; after weeks he learned that the activation he found was only due to the shaking of the cells and not the substance...)

Since changes can happen within milliseconds you should reflect this, if your outcome is questionable with the positive selection method, which may be OK depending on your experimental question, but I think any T-cell binding to anything gets strangely (non-specifically) activated. So maybe Dynabeads "untouched" or similar may work better for your approach.

You should do negative selection when possible to avoid the unwanted activation of the cells that can be observed when doing a positive selection such as anti-CD3. In my experience you can get up to 95% purification with negative selection avoiding T cell activation.

Do Negative selection!

I never worked with DYNAL beads but a colleague told me that at least for positive selected monocytes they cause activation of cells. In contrast beads from Miltenyi Biotech do not activate monocytes using positive selection, I think they offer reagents to select for T cells. Or go for negative selection as suggested above.

I m convinced with the answer of antonio

Hi David,

Firstly, I work for a company called STEMCELL Technologies and we offer column free immuno-magnetic separation systems for virtually any cell type. These are called EasySep and RoboSep and work in a similar principle to Dyna Beads but with different particle technology and magnets.

Now, in line with the comments above, if you are concerned about activation you should always choose negative selection. You do sacrifice slightly on purity and recovery but the bonus is you have 'untouched' cells. This becomes more important if you want to do functional studies post selection. For molecular work this may not be as much of a problem but it really depends on what your downstream experiments are. I would be happy to discuss this further with you as I am based in Melbourne. Separation of T Cells from PBMC's (using negative selection) can be done with our kits in as little as 25 minutes with 95-99% purity.

Feel free to message me if you would like more information

Hi David,

if you want to ensure that your cells do not get activated at all - go for the negative selection! When we tried human T cell enrichment with CD3 magnetic beads (StemCell), cells had a high purity but were highly activated as well (massive reduction of resazurine within few hours). Alternatively, you may try purification by nylon wool column (but it`s much more time-consuming).

Hi David. For all type of cells, binding with an antibody will activate the cognate receptor with the consequent molecular changes. Negative selection is the way to go. Surface attachment (to tubes and plates) also induce some changes, probably less prominent that antibody binding but still important, depending on what your testing is studying.

or go with MACS beads or cell sorting. Dynal beads can cause T cells to under go apoptosis

I have tried CD4 enrichment by Dyna beads before and found C-fos induced. Thus, it can partially activate cells.

Very interesting conversation. Can any of you provide a reference for these observations? Also, I'd be interested in understanding the industry, as opposed to academic, point of view regarding the significance of this issue.

Thanks for the constructive comments everyone.

Just to make things more interesting:-

What about positive selection by flow cytometry? Do people feel that using antibodies targeted to surface receptors suffers from the same problems?

I take the argument that engaging receptors induces activation, but do people really believe this would occur in the absence of sufficient cross-linking and co-activation?

cheers