Tissue Dispersion and Flow Cytometry for the Cellular Analysis of Wound Healing

Abstract

Injury induces a flux in the cellular composition of tissues as part of the wound healing response. There is no reliable and rapid method to quantify and characterize the cellular composition of the matrix-rich wound. Our aim was to develop a rapid method to quantify the cellular composition in wounds by tissue dispersion and flow cytometry. Age- and weight-matched mice were wounded on the dorsum using a 1.5 x 1.5 cm2 template, and the wounds were excised at predetermined time points. Tissues were dispersed into single-cell suspensions and labeled with antibodies to cell surfaces and intracellular antigens. Flow cytometry was performed to quantify the percentage of each cell population and cell death. We found that our tissue dispersion protocol resulted in low cell death (4%-6%) and very high yield (80-220 x 10(6) cells/g). Furthermore, cell surfaces and intracellular antigens were preserved to provide accurate identification of the different cell populations. With the appropriate modifications, this protocol is likely to be applicable for the viable retrieval and identification of cells from skin and other collagen-dense tissues.

Hoxa3 promotes the differentiation of hematopoietic progenitor cells into proangiogenic Gr-1+CD11b+ myeloid cells

“…Samples were analyzed or sorted using flow cytometry (BD FACSAria; BD Biosciences). Skin or wound tissue dispersion was performed essentially as described by Wilson et al 21 with modifications. In brief, freshly harvested skin or wound tissue was weighed and cut into 2-mm pieces followed by overnight incubation at 4°C in 5 mL/g Hank buffered salt solution containing 1 mg/mL dispase I (Sigma-Aldrich), 3% FBS, and 10 mg/mL G418 (Sigma-Aldrich).…”

Local Mesenchymal Stem/Progenitor Cells Are a Preferential Target for Initiation of Adult Soft Tissue Sarcomas Associated with p53 and Rb Deficiency

“…Plastic adherent dermal (PAD) cells were prepared from 6-to 7-week-old p53 loxP/loxP Rb loxP/loxP male transgenic mice according to a protocol by Wilson et al 30 with modifications. Briefly, euthanized mice were shaved and treated with Nair (Church & Dwight Co., Inc., Princeton, NJ) before skin removal.…”

Characterization of a murine keyhole limpet hemocyanin (KLH)-delayed-type hypersensitivity (DTH) model: Role for p38 kinase

“…We employed a protocol modified from that published by Wilson et al [39]. Briefly, ears were harvested and split into dorsal and ventral halves.…”