In Situ Single‐Cell Western Blot on Adherent Cell Culture.

Zhang, Yizhe; Naguro, Isao; Herr, Amy E.

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.

WESTERN immunoblotting; CELL culture; MITOGEN-activated protein kinases; CELL suspensions; PROTEIN analysis

Angewandte Chemie, 2019, Vol 131, Issue 39, p14067

0044-8249

Academic Journal

10.1002/ange.201906920