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Measurement of humoral factors secreted from cells has served as an indispensable method to monitor the states of a cell ensemble because humoral factors play crucial roles in cell–cell interaction and aptly reflect the states of individual cells. Although a cell ensemble consisting of a large number of cells has conventionally been the object of such measurements, recent advances in microfluidic technology together with highly sensitive immunoassays have enabled us to quantify secreted humoral factors even from individual cells in either a population or a temporal context. Many groups have reported various miniaturized platforms for immunoassays of proteins secreted from single cells. This review focuses on the current status of time-resolved assay platforms for protein secretion with single-cell resolution. We also discuss future perspectives of time-resolved immunoassays from the viewpoint of systems biology.
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Supplemental Video 1: Visualization of IL-1β release during pyroptosis induced by AIM2 inflammasome activation stimulated with LPS/poly(dA:dT) in peritoneal macrophages derived from SCAT1 mice, as discussed in Liu et al. (2014). The cells in the nanoliter well express the SCAT1 probe that abolishes FRET on the activation of Caspase-1, which is illustrated by a change from yellow to green here. Immediately after the Caspase-1 activation, the cells lose cell membrane integrity and become positive for SYTOX Orange, a dead cell staining reagent. Here, it is shown as coloring the nucleus purple. Immediately thereafter, cells release IL-1β extracellularly, here shown in cyan.
Supplemental Video 2: Tracking for the activity of a type 2 cytokine, IL-5, from mouse group 2 innate lymphoid cells (mILC2) isolated from fat associated lymphoid clusters in the mesentery using wide-field TIRFM based on an optical waveguide, as discussed Tanaka et al. (2017). Bright fields (left) and IL-5 secretion signal fluorescence (right, signal intensity is displayed in pseudo color) images in low-power (upper) or high-power (lower) observation. The trajectory from a cell tracking in the bright field is superimposed with magenta.
References
Liu T, Yamaguchi Y, Shirasaki Y, Shikada K, Yamagishi M, et al. 2014. Single-cell imaging of caspase-1 dynamics reveals an all-or-none inflammasome signaling response. Cell Rep. 8(4):974–82
Tanaka Y, Suzuki N, Mora K, Mizuno J, Shoji S, et al. 2017. Widefield real-time single-cell secretion imaging with optical waveguide technique. Paper presented at 19th International Conference on Solid-State Sensors, Actuators, and Microsystems (TRANSDUCERS), June 18–22, Kaohsiung, Taiwan. https://doi.org/10.1109/TRANSDUCERS.2017.7994363