Evaluation of two methods to isolate Schwann cells from murine sciatic nerve.

• SSchwann cells and macrophages play key roles in the aftermath of peripheral nerve injury. • Previous methods of Schwann cell isolation relied on pre-enrichment through cell culture, which alters phenotype. • This paper describes two methods for accurate separation of live Schwann cells and macrophages from injured murine nerve. • Live cells are sorted immediately after harvest, allowing gene expression analysis. • B6;D2-Tg(S100b-eGFP)1Wtj/J mice express GFP in Schwann cells and macrophages. Schwann cells (SC) and macrophages play key roles in the response to peripheral nerve injury (PNI). Accurate isolation of such cells is essential for further analyses that can lead to better understanding of the repair process after PNI. Separation of live SC from the injury site without culture enrichment is necessary for targeted gene expression analysis. Two flow cytometric techniques are presented for rapid enrichment of live SC and macrophages from injured murine peripheral nerve without the need for culture. SC were isolated by fluorescent activated cell sorting (FACS) using transgenic expression of eGFP in SC, or by exclusion of other cell types collected from the injury site. Gene expression analyses of peripheral nerve repair have commonly used whole nerve lysates. Isolating SC allows more accurate understanding of their specific role in repair. SC are commonly enriched from nerve by culture, however this changes gene expression patterns and limits the utility for transcriptomic analysis. The surface marker p75-NTR has variable expression in different SC phenotypes and during the course of injury and repair. Using p75-NTR for SC isolation might enrich only a subset of SC. More stably expressed lineage markers for SC are intracellular and not suitable for sorting for gene expression. The methods used here avoid the requirement for surface marker labeling of SC. Gene expression analysis of sorted cells from both methods showed successful enrichment of SC. Lineage markers such as Map1b, p75-NTR and S100b were enriched in the sorted SC population. SC sorting by eGFP expression showed improved enrichment, particularly of mature myelinating genes, although this could represent sampling of a subset of SC. [ABSTRACT FROM AUTHOR]