Dataset Information

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

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.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC6759404 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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

Zhang Yizhe Y Naguro Isao I Herr Amy E AE

Angewandte Chemie (International ed. in English) 20190821 39

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 copoly ...[more]

PMID: 31390130

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Dataset Information

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

Publications

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

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