Sean Lauber:Cytokine Stimulation of Cells: Difference between revisions
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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) | ||
# Split them | # 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. | ||
# | # 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) | ||
# Remove the media on the cells | # 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) |
Latest revision as of 06:58, 13 August 2013
- 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)
- 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.
- 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)
- Remove the media on the cells
- 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)
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