Table 1.

Examples of Commonly Used Techniques to Distinguish Tissue-Resident and Monocyte-Derived AMs

Species	Model	Explanation	Refs
Mouse	Ccr2 knockout (KO)	CCR2 is crucial for monocyte egress from the BM and recruitment from the blood into inflamed tissue. Therefore, in Ccr2 KO mice, changes in AMs can be attributed to the resident AMs, as monocyte recruitment is severely impaired.	[40,75]
	Busulfan chimeras using Ccr2 KO host	Busulfan is used to ablate BM cells, which are reconstituted with allogeneically marked BM cells (e.g., using CD45.1 or CD45.2 alloantigens). Donor-BM-derived monocytes are recruited into the CCR2-deficient host lung.	[10]
	Irradiation chimeras	In host mice, BM cells can be depleted through irradiation while preserving immune cells in the lung if the thorax is shielded. Reconstitution with marked BM cells allows tracing of BM-derived AMs.	[11,12,76]
	Parabiotic chimeras	Conjoined mice sharing blood flow develop a high level of chimerism of circulating monocytes. AMs that are marked with the alloantigen from the conjoined mouse must be monocyte derived.	[40,55]
	Fate mapping using Cre-loxP system	Granulocyte-monocyte progenitors express high amounts of Ms4a3. Monocytes express high amounts of Mx1, S100a4, and Flt3 as well as high and intermediate amounts of Cx3cr1. Resident AMs do not express these genes. Therefore, the promoter of the above genes is used to drive the expression of Cre recombinase that enables permanent induction of a fluorescent marker in monocyte-derived cells.	[3,4,16]
	Labeling with fluorescent antibody/dye	Intravenous injection of fluorescent antibody (e.g., against CD45) or dye (PKH26) labels monocytes and can be traced to monocyte-derived AMs. Dye (PKH26) administration into the lung labels phagocytic cells including resident AMs, so if it is administered before lung insult, newly incoming monocytes and monocyte-derived AMs will not be labeled. These are time-restricted labeling techniques.	[42,76]
Human	Lung transplant HLA mismatch	After lung transplant, donor resident AMs (bearing donor HLA) can be distinguished from host monocyte-derived AMs using mismatched HLA antigens.	[18]
	Lung transplant sex mismatch	In female recipients of a male-donor lung or vice versa, donor-lung-resident AMs can be distinguished from recipient-monocyte-derived cells using sex-specific transcripts [X-inactive specific transcript (XIST) for females and ribosomal protein S4 Y-linked 1 (RPS4Y1) for males] or fluorescence in situ hybridization (FISH) with X/Y probes.	[19,20]
	scRNA-seq	In scRNA-seq, AMs can cluster based on their expression of classical AM genes (potentially resident AMs) or peripheral monocyte-like genes (potentially monocyte-derived AMs). This method is correlative.	[77]