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, 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. The current study improves 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 using an enzyme-linked immunosorbent assay (ELISA) panel with human, adipose-derived stem cells stored for up to one month.

Mass‐Added Density Modulation for Sorting Cells Based on Differential Surface Protein Levels

“…For live-cell protein quantification, MADC-sorted cells from distinct density layers were lysed on ice for 30 min using radioimmunoprecipitation (RIPA, Santa Cruz Biotechnologies, Santa Cruz, CA) lysis buffer. For paraformaldehyde-fixed-cell protein tests, the previously described formaldehyde-fixed intracellular target-sorted antigen retrieval (FITSAR) method was used to isolate protein from MADC-sorted cells (18,19). Briefly, cells were suspended in lysis buffer consisting of 300 mM Tris hydrochloride (Sigma-Aldrich) with 2% sodium dodecyl sulfate (SDS; Thermo Fisher Scientific) and 2X protease/phosphatase inhibitor (Pierce™, Thermo Fisher Scientific) and boiled at 100 C for 35 min, followed by a 2-h incubation at 60 C. The samples were then spun down at 14,000g for 10 min before collecting the supernatant in a fresh 1.7 ml tube (Genesee Scientific).…”