Sean Lauber:Cytokine Stimulation of Cells

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  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 and count cells. Seed an appropriate number of cells into each well in normal media, and let the cells adhere for 1-2 hours.
  3. Prepare your stimulating media (often this is serum deprived) 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
  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