Table 1.

Technical Challenges for Discerning MNP Lineages in Retinal Degeneration.

Tools	Pros	Cons	Refs
Marker staining
Conventional markers, such as Iba-1, F4/80, Cd11b	Labels the majority of MNPs	Unable to distinguish microglia and MdCs Some markers not specific for MNPs	[2]
Microglial specific markers, such as P2ry12, Tmem119	Able to label specifically label microglia in steady state	May not distinguish microglia versus MdCs during neuroinflammation	[3,92,95]
Chimaeras
Bone marrow chimera	Full blood chimerism	Side effects of irradiation and transplantation, such as temporary disruption of BRB and myelomonocytic engraftment in the CNS	[2]
Bone marrow chimera with lead head shielding	Preservation of CNS in steady state	Full blood chimerism is not achievable	[11]
Circulating monocyte-labeling
Systemic 5- ethynyl-2′- deoxyuridine (EdU)	A traceable nucleotide that is integrated into the DNA of dividing cells Efficient labeling short-lived circulating monocytes and MdCs but not long-lived microglia in steady state. Nuclear stain, avoids confounding with (i) MNP auto-fluorescence and (ii) positivity due to transfer (phagocytosis) of reporter proteins	Require repeated injections, which limits use for short-term study but not chronic conditions. Microglia may replicate during degeneration, local injections necessary for distinction	[6]
Intravenously injected fluorescent tracers	A traceable fluorescent probe taken up by circulating monocytes and detectable in cells derived from these monocytes.	Require repeated injections, which limits it for short-term study but not chronic conditions.	[72]
Reporter mice
Cx3cr1GFP or Cx3cr1YFP	Heterozygotes are good for labeling MNPs Homozygotes are a full deletion of Cx3cr1 gene	Unable to distinguish microglia and MdCs, as both express Cx3cr1	[11,14,105]
Ccr2RFP	Heterozygotes are good for labeling classical monocytes Homozygotes are a full deletion of Ccr2 gene	Not good for labeling monocyte-derived macrophages, which lose Ccr2 expression Spares non-classical monocytes	[6,7,34]
Cx3cr1CreER R26fl-STOP-fl-reporter	Tamoxifen treatment induces the expression of reporter and labels microglia and MdCs The reporter expression in monocytes is ‘washed out’, as they are quickly replaced by non-labeled cells once tamoxifen decayed	Other long-lived tissue-resident macrophages that maintain Cx3cr1 expression may also be labeled	[9,11,34,84,85]
Depletion / recruitment inhibition studies
Csf1r antagonists	Efficient depletion of Csf1r dependent macrophages Easy for administration	Indistinguishably ablate microglia and MdCs Potential off-target effects on other tyrosine kinase receptors	[97,98,127]
Intravenous clodronate liposome	Efficient depletion of monocytes and their derivatives	Require repeated injections, which limits it for short-term study but not chronic conditions. Potential off-target effects	[6,98]
Cx3cr1CreER R26fl-STOP-fl-DTA	Efficient depletion of MNPs Not require diphtheria toxin	Indistinguishably depletes all Cx3cr1 expressing MNPs Require multiple tamoxifen administrations	[37,113]
Cx3cr1CreER R26fl-STOP-fl-DTR	Efficient depletion of microglia following ‘washout’	Require tamoxifen and diphtheria toxin administration Other long-lived tissue-resident macrophages may also be depleted	[3,84]
Ccl2- and Ccr2- deletion / inhibition	Efficiently inhibits exit of bone marrow classical monocyte into the blood	Does not inhibit non-classical monocyte-recruitment	[6–7, 73–80]