Figure 2. Protein expression of P2ry12, TMEM119 and FCRLS by engrafted monocytes.

CX3CR1^+/GFP::CCR2^+/RFP bone marrow chimeras were used to differentiate engrafted monocytes from embryonic microglia, followed by immunostaining and flow cytometry to assess expression of P2ry12, TMEM119, and FCRLS proteins. (A) P2ry12 is not expressed at day 1 and day 7 after monocyte infiltration into the retina, however 55% of GFP⁺ engrafted monocytes showed positive expression of P2ry12 at day 45. ***, P < 0.001. (B) Twenty-five percent of GFP⁺ peripheral monocytes (white arrow) expressed TMEM119 at day 1. By day 7, all GFP⁺ engrafted monocytes are TMEM119⁺. TMEM119 expression in engrafted monocytes is retained at day 45.**, P < 0.01; ***, P < 0.001. (C-D) A CX3CR1^+/GFP::CCR2^+/RFP bone marrow chimera was employed to assess Fcrls expression in monocytes and microglia by flow cytometry. BMT CX3CR1⁺ cells were labeled with a conjugated antibody against CX3CR1 (BV605 or APC) which allowed differentiation between embryonic microglia (BV605⁺GFP⁻ or APC⁺GFP⁻) and engrafted monocytes (BV605⁺GFP⁺ or APC⁺GFP⁺). Blood monocytes had no Fcrls expression (grey). One day after infiltration, BV605⁺GFP⁺ peripheral monocytes (purple) acquired strong Fcrls expression, which was comparable to naïve embryonic microglia. Fcrls expression was retained by engrafted monocytes sustained at day 7. At day 45, Fcrls expression was similar to embryonic microglia in the same injured tissue or to naïve microglia. *, P < 0.05. MFI: Median Fluorescence Intensity. Yellow arrows indicate GFP⁻ microglia and white arrows GFP⁺ engrafted monocytes. (A,B) Scare bar = 50 μm.