Fig. 3

Fate mapping showed that the retinal GFP^hi myeloid cells post-ONC were derived from microglia. a Tam induction of the RFP reporter in CX3CR1^YFP-CreER:R26^RFP mice labeled circulating CD11b⁺CD45^hi mononuclear cells, which substantially declined by 47 days. b Fundus photography showed that the retinal microglia were well labeled and expressed RFP at 70 days. c Flow cytometry showed that the microglia were induced to stably express RFP. (Left panels) An ONC-injured retina (ipsilateral) in mice lacking the CD11c-GFP transgene responded 7 days later with an increased number of microglia (YFP^hiRFP^hi) relative to the control (contralateral) retina. (Right panels) CX3CR1^YFP-CreER:R26^RFP:CD11c^GFP mice upregulated GFP on the YFP^hiRFP^hi microglia in the ipsilateral ONC retinas, accounting for a substantial portion of the increase in retinal myeloid cells. d Quantitation of the flow cytometry from c showing averaged results of groups of 3 to 6 mice. All GFP^hi cells were also RFP^hi and YFP^hi. Abbreviations: GFP^−/− = CX3CR1^YFP-CreER:R26^RFP mice. GFP^+/− = CX3CR1^YFP-CreER:R26^RFP:CD11c^GFP mice. Flow cytometry on retina included gating for viability, doublet exclusion and FSC/SSC prior to gating on CD45^medCD11b⁺Ly6G⁻ for expression of RFP, YFP and GFP. e Flatmount focused on the NFL of a retina post-ONC in a Tam-induced CX3CR1^YFP-CreER:R26^RFP:CD11c^GFP mouse showed that the GFP^hi cells were RFP^hi