Biological activity of cytokines and their concentrations are commonly measured by cellular proliferation of primary cell cultures or isolated cell lines that are dependent and/or responsive to a specific growth factor. Other aspects of biological activity of cytokines include induction of further cytokine secretion, induction of killing, antiviral activity, degranulation, cytotoxicity, chemotaxis, and promotion of colony formation. In vitro assays to measure all of these activities are available. Here, we discuss general protocols for setting up bioassays to measure:
Note: In setting up bioassays, the possibility of contaminating cytokines in the sample should be eliminated since many of the available indicator cell lines are responsive to more than one cytokine, and several cytokines share signaling receptors. Furthermore, the dependence and responsiveness of cell lines should be carefully ascertained since continuous culture of cells may alter their sensitivity to growth factors. Bacterial endotoxins are effective inducers of cytokines; hence, their presence in the bioassay cultures should be eliminated. In order to obtain statistically significant values, samples should be performed in triplicate wells.
Note: In interpreting bioassay data, note that many cytokines share receptor subunits and signaling second messengers and therefore could generate similar biological effect while being structurally different factors. False data generated due to the presence of other growth factors or soluble receptors can be further examined by blocking the effect of such reagents with specific antibodies.
The following tables summarize the conditions and reagents needed for human and mouse cytokine bioassays.
| Mouse Cytokine Bioassays Quick Guide | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Cytokine | Indicator Cells | Cell Density (x105) | Feeder Cytokine | Assay Medium FBS (%) | Incubation Time (hours) | Spec U/mg (10e) | Recomb. Cytokine Top Conc. (ng/ml) | ED50 (pg/ml) | Comments |
| IL-1b | D10S | 2 | None | 10 | 48 | 8 | 1 | 2 | |
| IL-2 | CTLL-2 (ATCC TIB-214) | 2 | hIL-2 @100U/ml | 10 | 24 | 7 | 10 | 80-300 | |
| IL-3 | MC/9 (ATCC CRL-8306), NFS-60 | 2 | mIL-3 @25U/ml | 10 | 48 | 7 | 10 | 80-300 | |
| IL-4 | CTLL-2 (ATCC TIB-214) | 2 | hIL-2 @100U/ml | 10 | 24 | 7 | 10 | 40-150 | |
| IL-6 | B9 | 2 | hIL-6 @50pg/ml | 10 | 48 | 8 | 0.1 | 3-12 | |
| IL-10 | MC/9 (ATCC CRL-8306) | 2 | mIL-3 @25U/ml | 10 | 48 | 7 | 10 | 150-600 | with mIL-4, 5pg/ml costimulation |
| IL-12 p70 | C20.4 | mIL-2 @100U/ml | 10 | 48 | 6 | 100 | 1000 | ||
| IL-15 | CTLL-2 (ATCC TIB-214) | 3 | IL-2 @100U/ml | 10 | 48 | 6 | 250 | 500-2000 | |
| IFNg | L929 (ATCC CCL-1) | 3.5 | None | 2 | 40 | 7 | 10 | 40-150 | EMCV @1x104 |
| TNFa | L929 (ATCC CCL-1) | 3.5 | None | 2 | 24 | 7 | 10 | 150-600 | Actinomycin D @2µg/ml final |
| GM-CSF | MC/9 (ATCC CRL-8306) | 2 | mIL-3 @ 25U/ml | 10 | 48 | 7 | 10 | 80-300 | |
| Human Cytokine Bioassays Quick Guide | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Cytokine | Indicator | Cell density (x105) | Feeder cytokine | Assay medium FBS (%) | Incubation time (hours) | Spec U/mg (10e) | Recomb. Cytokine Top Conc. (ng/ml) | ED50 (pg/ml) | Comments |
| IL-1a | D10S | 2 | None | 10 | 48 | 9 | 1 | 1 | |
| IL-1b | C3H/HEJ Thymocytes | 100 | None | 10 | 72 | 7 | 10 | 100 | |
| IL-2 | CTLL-2 (ATCC TIB-214) | 2 | hIL-2 @100U/ml | 10 | 24 | 7 | 10 | 80-300 | |
| IL-3 | TF-1 (ATCC CRL-2003) | 2 | hGM-CSF @100U/ml | 10 | 48 | 7 | 10 | 80-300 | |
| IL-4 | TF-1 (ATCC CRL-2003) | 2 | hGM-CSF @100U/ml | 10 | 48 | 7 | 10 | 80-300 | |
| IL-5 | TF-1 (ATCC CRL-2003) | 2 | hGM-CSF @100U/ml | 10 | 48 | 7 | 10 | 80-300 | |
| IL-6 | TF-1 (ATCC CRL-2003) | 2 | hGM-CSF @100U/ml | 10 | 48 | 7 | 0.1 | 80-300 | |
| IL-10 | MC/9 (ATCC CRL-8306) | 2 | mIL-3 @25U/ml | 10 | 48 | 6 | 10 | 1250 | costimulate with mIL-4 @ 5pg/ml |
| IL-12 p70 | hPBMC | None | 10 | 48 | 7 | 10 | 100 | ||
| IL-15 | CTLL-2 (ATCC TIB-214) | 3 | IL-2 @100U/ml | 10 | 48 | 6 | 1 | 500-2000 | |
| IFNg | A549 (ATCC CCL-185) | 3.5 | None | 2 | 40 | 7 | 10 | 300 | EMCV @ 4x104 |
| TNFa | L929 (ATCC CCL-1) | 3.5 | None | 2 | 24 | 6 | 10 | 500-2000 | Actinomycin D @2µg/ml |
| GM-CSF | TF-1 (ATCC CRL-2003) | 2 | hGM-CSF @ 100U/ml | 10 | 48 | 7 | 10 | 80-300 | |