Microfluidic Approach to Resolve Simultaneous and Sequential Cytokine Secretion of Individual Polyfunctional Cells.

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. This response involves numerous immune cells secreting various cytokines to orchestrate the immune reaction. However, the secretion dynamics, amounts, and co-occurrence of the different cytokines by various cell subtypes remain poorly understood due to a lack of appropriate tools to study them. Here, we describe a protocol using a microfluidic droplet platform that allows the time-resolved quantitative measurement of secretion dynamics for several cytokines in parallel on the single-cell level. This is enabled by the encapsulation of individual cells into microfluidic droplets together with a multiplexed immunoassay for parallel quantification of cytokine concentrations, their immobilization for dynamic fluorescent imaging, and the analysis of the respective images to derive secreted quantities and dynamics. The protocol describes the preparation of functionalized magnetic nanoparticles, calibration experiments, cell preparation, and the encapsulation of the cells and nanoparticles into droplets for fluorescent imaging and subsequent image and data analysis using the example of lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The presented platform identified distinct cytokine secretion behavior for single and co-secreting cells, characterizing the expected phenotypic heterogeneity in the measured cell sample. Furthermore, the modular nature of the assay allows its adaptation and application to study a variety of proteins, cytokines, and cell samples, potentially leading to a deeper understanding of the interplay between different immune cell types and the role of the different cytokines secreted dynamically to shape the tightly regulated immune response. These new insights could be particularly interesting in the studies of immune dysregulations or in identifying target populations in therapy and drug development.

Simultaneous Quantification of Commonly Used Counter Ions in Peptides and Active Pharmaceutical Ingredients by Mixed Mode Chromatography and Evaporative Light Scattering Detection.

In academia and industry, the analysis of counter ions in small molecules and synthetic peptides represents a great challenge. Due to the frequent use of salt forms and the application of a wider range of counter ions in pharmaceutically used substances, simple and generic methods for quantification are required. Especially, the analysis of trifluoracetic acid (TFA) in synthetic peptides is of high interest. Quantification of TFA is needed to assess the content and safety of synthetic peptides and for the interpretation of functional assay results, respectively. In here, a full quantitative mixed mode high performance liquid chromatography based method coupled to evaporative light scattering detection is presented. Finally, 14 positively and negatively charged counter ions were simultaneously quantified within 30 minutes. The method was validated in terms of specificity, accuracy, precision, limit of quantification, sample stability and carry over as proposed by the International Council of Harmonization. In order to prove the applicability of the procedure, small molecules reference substances and synthetic peptides were analyzed, respectively. The obtained results indicated a successful determination of counter ions in small molecules and differences to expected concentrations of prepared peptide solutions. Furthermore, an unexpectedly high content of sodium was observed for synthetic peptides.