Sean Lauber:Cytokine Stimulation of Cells

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Current revision (08:58, 13 August 2013) (view source)
 
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# 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)
# 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)
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# 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)
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# 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.
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# 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)
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# 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)
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# Remove the media on the cells and wash 2x with sterile PBS
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# Remove the media on the cells
# Add the stimulating media and incubate for the desired time
# Add the stimulating media and incubate for the desired time
# Colect the supernatant and spin it down to remove any cells that might interfere with an ELISA (if you plan on using an ELISA)
# Colect the supernatant and spin it down to remove any cells that might interfere with an ELISA (if you plan on using an ELISA)

Current revision

  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

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