Tissue dispersion and flow cytometry for the cellular analysis of wound healing.
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.
Wilson
Lynne
L
Puget Sound Health Care System & University of Washington Medical Center, Seattle, USA.
Fathke
Carrie
C
Isik
Frank
F
D000818:Animals; D002470:Cell Survival; D020405:Dermis / Q000166:cytology* / Q000293:injuries; D000078404:Epidermal Cells; D004817:Epidermis / Q000293:injuries; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D015203:Reproducibility of Results; D014945:Wound Healing / Q000235:genetics*
D016326:Extracellular Matrix Proteins