T helper differentiation protocols can be really tricky!
Can you please provide a detailed protocol? what type of cells (mouse or human and if mouse what strain of mice)
How are you activiating? Are the cells really blasting?
How long are you differentiating?
How much cytokines are added (are you adding neutralizing abs)?
When are you restimulating? and how? are you adding golgi block or goldi stop?
How long are you restimulating?
I usually use NOD CD4+CD62L+ cells sorted by MACS. I usually pool the T cells from LN and Spleen. I use APCs. I then add various combinations of cytokines to the cells the same day that I plate the cells out. I have tried just with aCD3, or aCD3+TGF, aCD3+TGF+IL-6, aCD3+TGF+IL-6+IL-23, aCD3+TGF+IL-6+IL-23+IL-1b. I then leave the cells for 4 days. On the 4th day I restimulate the cells for about 4 hours by adding PMA and ionomycin. Depending on whether I am doing intracellular staining or ELISA, I add or do not add golgiplug (BD Biosciences). I don't add neutralizing antibodies. The cells seem to be blasting ok. Hope I answered all your questions.
I was also wondering, along with another poster, the specifics of handling the plating and medium changes.
I have read protocols where medium changes and cytokine addition occur on day 2 and 4 (day 0 being plating day), but I cant't figure out how this can be managed on a plate larger than 96 wells without losing cells (and beads) in the spins and generally creating a lot of cell handling mess.
I was hoping to do this in a 48 or 24 well tray, because by the time I get through with intracellular staining and all the spins, etc, I loose a lot of cells if my source is just 1 well from a -96 well tray. Generally, I split the 1 well on day 4, then recombine it for the intracellular staining. It's still not enough. Seeing the pellet get smaller and smaller with each successive spin during permeabilization and staining is depressing!
The 24-well protocol referenced in the previous post is a 3-day with no re-feed. I am doing a 5-day human cell protocol, so I need to re-feed and split. Any suggestions?
Thanks so much, I appreciate the time taken by folks to help out.
Edited by tar bal, 27 May 2010 - 08:33 AM.