OKT3 and anti-CD28 to stimulate Jurkat T-cells - (Sep/09/2009 )

Hello,

I am trying to use OKT3 and anti-CD28 antibodies to stimulate human T-cells. The majority of protocols and other information I have found describes how to do this with PBMCs, but I will be using the Jurkat T-cell line because we will be stimulating the cells in order to collect RNA and need a clean population. A few people that I have spoken to regarding this issue have mentioned that we may need to crosslink the antibodies in some way, pre-coat the plates with the OKT3, or use coated Dynabeads. Does anybody have any insight or suggestions as to how I can successfully activate this cell line using the OKT3 and anti-CD28 antibodies? Can anyone point me in the direction of a protocol that has been successful?

Thank you.

Just a side note, not all Jurkat lines respond to antibody cross-linking...

You can do this in a few ways...
1. Coat the plate with your antibodies. Coat in a minimal amount of PBS (don't use media or PBS with BSA in it). Coating overnight at 4 degrees is the best option. You can also do room temp for at least 2 hours or 37 degrees for 1 hour.

2.You can use an anti-mouse IgG2a antibody (I think this is the OKT3 clone) and an antibody against the CD28 antibody. This will cause crosslinking also.