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Biotechniques. 2017 Dec 1;63(6):275-280. doi: 10.2144/000114620.

Processing fixed and stored adipose-derived stem cells for quantitative protein array assays.

Author information

1
Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI.
2
Center for Biomedical Engineering, Brown University, Providence, RI.
3
School of Engineering, Brown University, Providence, RI.
4
Department of Orthopaedics, Brown University, Providence, RI.

Abstract

Accurately characterizing cellular subpopulations is essential for elucidating the mechanisms underlying normal and pathological biology. Isolation of specific cell types can be accomplished by labeling unique cell-associated proteins with fluorescent antibodies. Cell fixation is commonly used to prepare these samples and allow for long-term storage, but this poses challenges for subsequent protein analysis. We previously established the FITSAR (formaldehyde-fixed intracellular target-sorted antigen retrieval) method, in which protein can be isolated and characterized from fixed, enriched cell subpopulations. Here, we improve on this method by allowing compatibility with highly sensitive multiplex protein arrays and demonstrating applicability to long-term stored samples. Feasibility experiments demonstrated parallel detection of cell adhesion molecules (CAMs) using an enzyme-linked immunosorbent assay (ELISA) panel with human adipose-derived stem cells (ASCs) stored for up to 1 month.

KEYWORDS:

FITSAR; multiplex ELISA; paraformaldehyde; protein characterization

PMID:
29235974
PMCID:
PMC5731247
DOI:
10.2144/000114620
[Indexed for MEDLINE]
Free PMC Article

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