In Situ Single-Cell Western Blot on Adherent Cell Culture

Angewandte Chemie
Yizhe ZhangAmy E Herr

Abstract

Integrating 2D culture of adherent mammalian cells with single-cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single-cell isolation and protein analysis. To assay HeLa cells from an attached starting state, we culture adherent cells in fibronectin-functionalized microwells formed in a thin layer of polyacrylamide gel. To integrate the culture, lysis, and assay workflow, we introduce a one-step copolymerization process that creates protein-decorated microwells. After single-cell culture, we lyse each cell in the microwell and perform western blotting on each resultant lysate. We observe cell spreading after overnight microwell-based culture. scWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions. We validate the in situ scWB with slab-gel western blot, while revealing cell-to-cell heterogeneity in stress responses.

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Citations

Dec 6, 2020·Nature Communications·Samantha M GristAmy E Herr
Mar 4, 2021·Trends in Biochemical Sciences·Luke F Vistain, Savaş Tay
Apr 14, 2021·Biochemistry and Molecular Biology Education : a Bimonthly Publication of the International Union of Biochemistry and Molecular Biology·Gholam Hossein MeftahiZohreh Jangravi
Feb 6, 2020·Analytical Chemistry·Yuri M ShlyapnikovElena A Shlyapnikova

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