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).

Thanks for the clarification. High levels of serum may interfere with readings but as long as you have the appropriate controls, this may not be an issue. Since you have human serum and cytokines in your culture media, the negative control is a bit tricky. What are your samples? If you are just assaying cytokines during DC differentiation, use your D0 samples as your baseline. Theoretically, they should not be secreting much. If you want to assay cytokines in response to a specific treatment (e.g. LPS), use untreated cells (cultured in media containing human serum and cytokines) as your control.

Another best practice is to not measure cytokines that you are adding to the culture. This means that if you are adding GM-CSF to differentiate DCs, you should not be using GM-CSF as a read-out. Any difference you observe could be due to several reasons and it is almost impossible to figure out which one it is.