A Streamlined Approach to Quantify Inflammatory Cytokines from Serum and Skin

Cytokine release syndrome (CRS) is a systemic inflammatory condition that arises when immune cells such as natural killer (NK) cells, macrophages, and dendritic cells become activated, leading to the production of inflammatory cytokines. CRS can be triggered by treatments like CAR-T cell therapy, infections causing sepsis, and other factors. This excessive cytokine release creates a feedback loop that intensifies the inflammation, potentially resulting in severe tissue damage1 . 

A key part to understanding CRS and how to reduce its detrimental effects is centered around immunology research where an efficient and consistent quantification of local immune mediators is crucial. The first step to quantifying cytokines from tissue is sample lysis, a method where individual cells comprising the tissue are lysed releasing all intracellular biomolecules. While a variety of methods exist for sample lysis, efficiency is key here, as sample homogenization can become a time sink if using less than optimal methods. 

Sonication, for example, is a tried-and-true technology that works by delivering ultrasonic frequencies to a sample using a stainless-steel horn. Typically, this process is completed one sample at a time and cleaning steps must be performed in between samples to remove residual sample material from the ultrasonic processing horn. In contrast, bead mill homogenization using the Omni Bead Ruptor Elite™ bead mill homogenizer offers a robust solution to tissue homogenization by tackling multiple samples at a time in a semi-automated, repeatable and time-saving manner generating suitable lysates for cytokine quantification.

By enabling researchers to simultaneously quantify inflammatory cytokines and biomarkers, the cellular effects of cascading cytokine release can be well studied and understood at the lab bench, propelling forward areas of immunology disease research and development of immunotherapies. One approach is to analyze the cytokine profile from serum and plasma samples, however, often the concentrations are low if immune response is limited to tissues. Thus, investigating the cytokine release in tissue to understand the microenvironment is becoming more important in various research areas. Herein, we outline a workflow for tissue lysate preparation and simultaneous quantification of 13 key biomarkers relevant to CRS using the LEGENDplex™ Mouse Cytokine Release Syndrome panel to quantify IFN-γ, IL-10, CCL4, IFN-α, CXCL9, CXCL10, TNF-α, IL-6, VEGF, IL-4, CCL3, CCL2, and GM-CSF (Figure 1). This assay panel provides higher detection sensitivity and broader dynamic range than traditional ELISA methods by utilizing fluorescence-encoded beads suitable for use on various flow cytometers.

Figure 6. Level of CCL4, CCL2 and CCL3 quantified from skin tissue prepared using a sonicator or bead mill homogenizer from LPS and PBS groups.