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Front. Immunol. | doi: 10.3389/fimmu.2018.00347

Quantitative methods for measuring repair rates and innate immune cell responses in wounded mouse skin

 Zhi Li1, 2, Elizabeth Gothard2,  Mark C. Coles2, 3* and  Carrie Ambler1*
  • 1Durham University, United Kingdom
  • 2University of York, United Kingdom
  • 3University of Oxford, United Kingdom

In skin wounds, innate immune cells clear up tissue debris and microbial contamination, and also secrete cytokines and other growth factors that impact repair process such as re-epithelialization and wound closure. After injury, there is a rapid influx and efflux of immune cells at wound sites, yet the function of each innate cell population in skin repair is still under investigation. Flow cytometry is a valuable research tool for detecting and quantifying immune cells, however in mouse back skin, the difficulty in extracting immune cells from small area of skin due to tissue complexity has made cytometric analysis an underutilized tool. Here in this paper, we provide detailed methods on the digestion of lesion-specific skin without disrupting antigen expression followed by multiplex cell staining that allows for identification of seven innate immune populations, including rare subsets such as group 3 innate lymphoid cells (ILC3s), by flow cytometry analysis. Further, when studying the functions of immune cells to tissue repair an important metric to monitor is size of the wound opening. Normal wounds close steadily albeit at non-linear rates, while slow or stalled wound closure can indicate an underlying problem with the repair process. Calliper measurements are difficult and time-consuming to obtain and can require repeated sedation of experimental animals. We provide advanced methods for measuring of wound openness; digital 3D image capture and semi-automated image processing that allows for unbiased, reliable measurements that can be taken repeatedly over time.

Keywords: flow cytometry methods, innate immunity, Skin, wound, 3D imaging

Received: 10 Nov 2017; Accepted: 07 Feb 2018.

Edited by:

Xiaoquan Rao, Case Western Reserve University, United States

Reviewed by:

Liang Cao, Northwestern University, United States
Aguang Dai, University of Texas at Austin, United States
Juli Bai, University of Texas Health Science Center San Antonio, United States  

Copyright: © 2018 Li, Gothard, Coles and Ambler. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Prof. Mark C. Coles, University of York, York, United Kingdom, mark.coles@kennedy.ox.ac.uk
Dr. Carrie Ambler, Durham University, Durham, United Kingdom, c.a.ambler@durham.ac.uk