T cell activation - (Mar/06/2006 )
Hi!
I want to activate my T cells but i'm wondering what's the difference between ConA, PHA+IL2, PMA....??? 
I actually wants a good T cell activation but as I want to do FACS afterwards I'd like them not to be clumpy.So wich way would be the best?
Thanks 
ConA is a lectin that binds to and non-specifically activates T cells
PHA and PMA are phorbol esters compounds that activate multiple proliferative signalling pathways in cells
IL-2 is a T cell proliferating interleukin
We often activate T cells with most of these methods. It sort of depends on the cell type, which works the best for good activation. I would try multiple methods in at first and see whats best.
T cells upregulate adhesion molecules upon acitvation. I think this is supposed to allow them to exit the vasculature into the tissues or something. Anyways, in culture, activated T cells should actually look clumpy as you describe I think. If you're worried that this might affect the FACS staining I suppose you could try using a sterile cell strainer before the staining.
Good luck
Thanks for the quick reply 
I have to work on PBMC's from Ficoll and I decided to try either ConA and PHA to stimulate them...For either one I was wondering 
I want to optimize the membrane expression of my protein at my t cell membrane (that's the reason why i activate them) and the articles that i read tells that it needs a 3 day activation to have a maximum expression...
But the question is do I leave the PHA or the ConA during ?? or do i wash it before?they don't say a word about it 
And i presume i'll have to add IL-2 for them not to die but I long after?Right after adding my PHA?I don't know i'm trying to read as much articles as i can but they don't gives such details.
Thanks in advance if u can help me with this.
PHA and PMA are phorbol esters compounds that activate multiple proliferative signalling pathways in cells
IL-2 is a T cell proliferating interleukin
We often activate T cells with most of these methods. It sort of depends on the cell type, which works the best for good activation. I would try multiple methods in at first and see whats best.
T cells upregulate adhesion molecules upon acitvation. I think this is supposed to allow them to exit the vasculature into the tissues or something. Anyways, in culture, activated T cells should actually look clumpy as you describe I think. If you're worried that this might affect the FACS staining I suppose you could try using a sterile cell strainer before the staining.
Good luck
Can't you just pipet up and down to declump them? Just wondering.
Yes I would leave the ConA or PHA in the media the whole incubation
yeah of course you can pipette them up and down. The beauty of the strainer is that you can turn up the acquisition rate on the FACS machine SUPER high without fear of clogging the machine, which is useful for sorting small cell populations and stuff. Just a trick I learned from our FACS operator.
yeah of course you can pipette them up and down. The beauty of the strainer is that you can turn up the acquisition rate on the FACS machine SUPER high without fear of clogging the machine, which is useful for sorting small cell populations and stuff. Just a trick I learned from our FACS operator.
I did pipet up and down and it seems to works (just looked in the microscope)i'll see the results withe the FACS this afternoon.
But i realised that a lot of cells (even after pipetting up and down) remained stuck in the bottom of my plate (u-bottom plates) and I was wondering maybe you could give me an idea to "un-stuck" them from the bottom of the plate?
hi,
to unstuck the cells try PBD/EDTA 2mM for 10min, then hit the umpty plate firmly and recover the detached cells in PBS; it work fine for dendritic cells
Seb