Sean Lauber:Cytokine Stimulation of Cells

1. Grow your cells and split a couple of times to bring them to a normal state (thawing them a stimulating immediately is not a good idea because the cells are likely stressed)
2. Split them so they will be at an appropriate confluency for treatment the following morning (if you need to treat them for 2 days, then split them so that the following morning you'd have to split them in 2 days - again this is done to minimize stress)
3. The following morning prepare your stimulating media in an appropriate volume for treatment (for example, if you have 100 ul on your cells normally and you have 9 wells to treat, prepare your stimulating media at the appropriate concentration with enough volume for 9 * 10% (pipetting error) wells - if you're using a multichannel, increase the volume appropriately)
4. Remove the media on the cells and wash 2x with sterile PBS
5. Add the stimulating media and incubate for the desired time
6. Colect the supernatant and spin it down to remove any cells that might interfere with an ELISA (if you plan on using an ELISA)

Some common concentrations used:

 LPS:     100 ng/ml
 mIL-6    10 ng/ml
 mTNF-a   10 ng/ml
 mIL-4    10 ng/ml
 mIL-13   10 ng/ml
 mIFN-g   10 ng/ml
 hTGF-b   5 ng/ml

Download the template to assist in figuring out what volumes to use