Digital microfluidic immunocytochemistry in single cells

• Published in: Nature communications Vol. 6; no. 1; p. 7513
• Main Authors: Ng, Alphonsus H. C., Chamberlain, M. Dean, Situ, Haozhong, Lee, Victor, Wheeler, Aaron R.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 24.06.2015; Nature Publishing Group
• Subjects: 13/1; 13/106; 13/56; 13/62; 631/1647/1407; 631/1647/664/1257; 631/80/2373; 631/80/86; 96/1; 96/63; 96/95; Animals; Automation; Cell culture; Cell Culture Techniques; Cell Line; Downstream effects; Fibroblasts; Fibroblasts - metabolism; Flow cytometry; Growth factors; Humanities and Social Sciences; Humans; Immunohistochemistry - instrumentation; Immunohistochemistry - methods; Interrogation; Kinases; Ligands; MCF-7 Cells; Mice; Microfluidic Analytical Techniques - instrumentation; Microfluidic Analytical Techniques - methods; Microfluidics; multidisciplinary; NIH 3T3 Cells; Phosphorylation; Platelet-derived growth factor; Platelet-Derived Growth Factor - metabolism; Proteins; Proto-Oncogene Proteins c-akt - metabolism; Reagents; Science; Science (multidisciplinary); Shear stress; Signal Transduction; Signaling; Stimulation; Time dependence
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms8513

We report a new technique called Digital microfluidic Immunocytochemistry in Single Cells (DISC). DISC automates protocols for cell culture, stimulation and immunocytochemistry, enabling the interrogation of protein phosphorylation on pulsing with stimulus for as little as 3 s. DISC was used to probe the phosphorylation states of platelet-derived growth factor receptor (PDGFR) and the downstream signalling protein, Akt, to evaluate concentration- and time-dependent effects of stimulation. The high time resolution of the technique allowed for surprising new observations—for example, a 10 s pulse stimulus of a low concentration of PDGF is sufficient to cause >30% of adherent fibroblasts to commit to Akt activation. With the ability to quantitatively probe signalling events with high time resolution at the single-cell level, we propose that DISC may be an important new technique for a wide range of applications, especially for screening signalling responses of a heterogeneous cell population. The ability to measure signalling responses in single cells following short pulses of stimulus would shed insight into temporal thresholds for cell activation. Here the authors introduce a microfluidic platform that allows downstream phosphorylation cascades to be observed following as little as one second of stimulus exposure.