Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap
Yacine Bounab1,2, Klaus Eyer3,4, Sophie Dixneuf5
1BIOASTER Technology Research Institute, Lyon, France.
Insights
A new microfluidic platform, DropMap, enables dynamic, quantitative analysis of single immune cells and their secreted proteins like cytokines and antibodies, overcoming limitations of current methods.
Area of Science:
- Immunology
- Microfluidics
- Cell Biology
Background:
- Current methods for immune response characterization lack quantitative, dynamic analysis of single cells and secreted proteins.
- Existing techniques like ELISPOT, flow cytometry, CyTOF, and single-cell sequencing provide only end-point measurements.
- There is a need for systems that can simultaneously measure secreted protein kinetics and cellular phenotypes.
Purpose of the Study:
- To introduce and validate DropMap, a microfluidic platform for high-throughput, quantitative, and dynamic analysis of single immune cells.
- To demonstrate the platform's capability in measuring secretion kinetics of cytokines and antibodies alongside cellular characteristics.
- To showcase DropMap's application in profiling immune responses in disease contexts.
Main Methods:
- Development of a microfluidic platform (DropMap) for compartmentalizing single cells in 50-pL droplets.
- Integration of fluorescence microscopy with a paramagnetic nanoparticle-based immunoassay for quantitative protein detection.
- Simultaneous measurement of secreted protein kinetics, endocytosis activity, viability, and cell-surface markers.
Main Results:
- DropMap enables simultaneous measurement of secretion kinetics and multiple cellular phenotypes from tens of thousands of single immune cells.
- The protocol is robust, with a total assay time of 8-10 hours.
- Demonstrated applications include profiling tumor necrosis factor-α (TNF-α) secretion in septic shock patients, analyzing T cell cytokine secretion rates, and measuring B cell antibody affinity.
Conclusions:
- DropMap offers a significant advancement for characterizing immune responses by enabling quantitative, dynamic, single-cell analysis of secreted proteins.
- The platform overcomes limitations of existing end-point measurement techniques.
- DropMap has broad applicability for studying immune cell function in health and disease.
Abstract:
Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8-10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.
