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

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
• protein characterization
• paraformaldehyde
• multiplex ELISA