I want to track relative increases/decreases in cytokine during iDC and mDC cultures. I was thinking I could use a cytokine array kit as they are easy to use and off the shelf/don't take any development. Then I could see which cytokines are 'important' and develop ELISA for those. My questions are;
1. Will the kit be sensitive enough to detect changes in cytokine concentration without stimulation?
2. How does human serum effect result?
3. Can you recommend a good kit?
4. Do I need any specialist equipment?
5. What is usually used for positive and negative controls?
6. We have cytokines in our media. Can the kit be used to track whether they are in excess?
Thanks
Nicola
1. Most kits have a detection limit of 7.5 pg/mL and if your unstimulated cells are secreting cytokines at levels >LOD, then the answer is ‘yes’.
2. Some of the kits out there are compatible with human serum. The product sheet would say whether or not the kit works with serum samples.
3. Depend on your experimental setup, the cytokines you want to measure and the equipment you have access to. I have experience using all the different systems in answer #4 and they all worked well.
4. Depends on the kit you are using. BD CBA, BioLegend LegendPlex and other bead based systems need access to a flow cytometer. Luminex kits (ThermoFisher) need access to a Luminex machine. R&D proteome profiler kits are based on western blots and so any lab that has western blotting apparatus can do this. Qiagen’s multi-analyte ELISA kits are ELISA based and all you need is a spectrophotometer that can measure absorbance. Bead based systems are replacements for traditional ELISAs. The products from R&D and Qiagen are not as accurate and these are meant to be preliminary assays that you need to follow up with individual ELISAs.
5. Negative control- unstimulated cells; positive control- cells stimulated with a polyclonal stimulus like LPS, PMA, ConA, PHA, PMA + ionomycin (or something that you know should stimulate your cells).
6. Not sure what you mean by ‘in excess’. All kits have a linear range (e.g. 7.5-1000 pg/mL) and as long as your cytokine levels are within this range, the measurements are accurate. If they are above this range, you would have to dilute your samples appropriately (e.g. at least 1:2 of a sample that contains 2000 pg/mL of the cytokine) before you run them.
1. Most kits have a detection limit of 7.5 pg/mL and if your unstimulated cells are secreting cytokines at levels >LOD, then the answer is ‘yes’.
2. Some of the kits out there are compatible with human serum. The product sheet would say whether or not the kit works with serum samples.
3. Depend on your experimental setup, the cytokines you want to measure and the equipment you have access to. I have experience using all the different systems in answer #4 and they all worked well.
4. Depends on the kit you are using. BD CBA, BioLegend LegendPlex and other bead based systems need access to a flow cytometer. Luminex kits (ThermoFisher) need access to a Luminex machine. R&D proteome profiler kits are based on western blots and so any lab that has western blotting apparatus can do this. Qiagen’s multi-analyte ELISA kits are ELISA based and all you need is a spectrophotometer that can measure absorbance. Bead based systems are replacements for traditional ELISAs. The products from R&D and Qiagen are not as accurate and these are meant to be preliminary assays that you need to follow up with individual ELISAs.
5. Negative control- unstimulated cells; positive control- cells stimulated with a polyclonal stimulus like LPS, PMA, ConA, PHA, PMA + ionomycin (or something that you know should stimulate your cells).
6. Not sure what you mean by ‘in excess’. All kits have a linear range (e.g. 7.5-1000 pg/mL) and as long as your cytokine levels are within this range, the measurements are accurate. If they are above this range, you would have to dilute your samples appropriately (e.g. at least 1:2 of a sample that contains 2000 pg/mL of the cytokine) before you run them.
Thanks for your detailed answer it's very useful!
I will be using cell culture supernatant. For questions 2 and 6 my issue is that we add human serum and cytokines to our cell culture media so they will interfere with the read-out?
We use BioPlex from biorad and as Julie Joseph suggested the limit of detection is in the range of 5 pg/ml.
For the questions 2 and 6 the way to nullify/normalize the preexisting cytokines is to use a control which has the same composition in terms of FBS and media.
You may use a separate control or you may use the media containing FBS as diluent for the standards and the blank should be only the diluent (media+ FBS).
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