Flow Cytometry Rapidly Determines Specific Interleukin-6 Activity

The measurement of the specific activity of a given cytokine often requires a time-consuming and tedious bioassay based upon its effect on the proliferation, chemotaxis, or toxicity of a factor-dependent cell line.

The determination of the specific activity of the cytokine interlukin-6 (IL-6) was accomplished by ascertaining the dose–response curve of the phosphorylation of a signaling protein that can be quantified by flow cytometry.

Immunologists at Héma-Québec (Montreal, QC, Canada) used the IL-6 model, as an approach for the determination of the specific activity of cytokines by determining the dose–response curve of the phosphorylation of the signaling protein Stat-3 pY705 for IL-6, in a responsive cell line subjected to different concentrations of the cytokine. The phosphorylation of the protein is quantified by flow cytometry. Since the signaling cascade only takes a few minutes to achieve following linking of the cytokine to its receptor, the whole procedure can be done in a few hours.

The scientists used the cell line 7TD1 (DSMZ, German Collection of Microorganisms and Cell Cultures GmbH; Braunschweig, Germany), which is an IL-6 dependent cell line. The cells were cultured in RPMI-1640 containing 5% fetal bovine serum (FBS) and 10 ng/mL of IL-6. To speed up flow cytometry, the samples were fluorescently barcoded by adding Pacific Blue and Alexa Fluor 488 to allow multiplex analysis. After processing the cells were analyzed on a Partec CyFlow ML flow cytometer (Swedesboro, NJ, USA).

The scientists were able to determine and compare the specific activity of commercial lots of IL-6 using the flow cytometry method. They observed significant differences both from lots from different manufacturers and lots from a same manufacturer, with an eightfold difference between the specific activity of the highest and lowest one. This demonstrated that in studies requiring accurate cytokine concentrations, determining the specific activity for each lot should be a crucial step in order to get reproducible results.

Higher levels of IL-6 have been observed in the blood of advanced or metastatic cancer patients and are associated with the development of encephalitis in children. The study was published on October 5, 2014, in the Journal of Immunological Methods.

Related Links:
Héma-Québec
German Collection of Microorganisms and Cell Cultures GmbH 
Partec