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Merck

Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap.

Nature protocols (2020-08-14)
Yacine Bounab, Klaus Eyer, Sophie Dixneuf, Magda Rybczynska, Cécile Chauvel, Maxime Mistretta, Trang Tran, Nathan Aymerich, Guilhem Chenon, Jean-François Llitjos, Fabienne Venet, Guillaume Monneret, Iain A Gillespie, Pierre Cortez, Virginie Moucadel, Alexandre Pachot, Alain Troesch, Philippe Leissner, Julien Textoris, Jérôme Bibette, Cyril Guyard, Jean Baudry, Andrew D Griffiths, Christophe Védrine
ZUSAMMENFASSUNG

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.

MATERIALIEN
Produktnummer
Marke
Produktbeschreibung

Sigma-Aldrich
PMA, for use in molecular biology applications, ≥99% (HPLC)
Sigma-Aldrich
Albumin human, Cellastim S, recombinant, expressed in rice, lyophilized powder, suitable for cell culture, low endotoxin, ≥96% (SDS-PAGE)
Sigma-Aldrich
Lipopolysaccharide aus Escherichia coli O55:B5, purified by gel-filtration chromatography
Sigma-Aldrich
Ethanolamin, ≥98%