**5. Conclusions**

Flow cytometry is an e ffective and high-throughput method to analyze and quantify infiltrated leukocyte subpopulations in muscle tissue. If necessary, it can be supplemented by IHC studies on the localization of those leukocytes.

The protocol includes some limitations. Inadequate perfusion of the animals may be associated with residuals of leukocyte in vessels, resulting in inconsistent data. Poor enzymatic muscle digestion may lower the cell yield. In addition, cell preparation that involves washing and centrifugation might lead to cell loss of an unknown extent.

However, when accurately performed, flow cytometry is an elegant method that can be used to complement results obtained from histological analysis, especially those pertaining to cell di fferentiation and cell counting. Further, flow cytometric analysis gives the possibility to calculate the absolute number of leukocytes per mg muscle tissue by the addition of counting beads to the cell suspension. With the increasing functions of flow cytometers, the further development of tissue extraction methods as well as the capacity to stain various markers on cell surfaces and in the cell interior, the application spectrum of flow cytometry is extremely broad.

**Author Contributions:** Conceptualization, K.K., N.S., S.B., S.R. and E.D.; methodology, K.K., N.S., S.R. and L.R.; validation, K.K., N.S., S.R., L.R. and B.U.S.; investigation, K.K., N.S., S.R. and L.R.; data curation, K.K.; writing—original draft preparation, K.K., N.S., S.R., and S.B.; writing—review and editing, N.S., S.R., A.-K.K., M.L., L.R., B.U.S. and E.D.; supervision, B.U.S., E.D.; project administration, B.U.S. and E.D.; funding acquisition, B.U.S. and E.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Lehre@LMU program (A.-K.K., E.D.) and the Förderprogramm für Forschung und Lehre (FöFoLe) (S.B., E.D.) from the LMU Munich. The work in the Schraml lab was supported by the German Research Foundation (Emmy Noether grant: Schr 1444/1-1; and project number 360372040—SFB 1335—project 8, to Schraml) and the European Research Council (ERC-2016-STG-715182).

**Conflicts of Interest:** The authors declare no conflict of interest.
