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. 2016 Nov 24;10:526. doi: 10.3389/fnins.2016.00526

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Copyright © 2016 Breen, Anderson, Steele, Calkins, Bosco and Vetter.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Amoeboid Iba1+ cells are peripherally derived macrophages. (A) Amoeboid Iba1+ cells localize to the neurofibrillary layer. Volume view of a DBA/2J retinal whole mount stained for the axonal marker pNF (red) and the microglia/macrophage marker Iba1 (green). Orthogonal views shown below and to the right. Amoeboid Iba1+ cells showed non-specific reactivity with the donkey anti-mouse secondary antibody used to detect pNF (not shown). (B) Single z-plane (0.8 μm) from a control DBA/2J retinal whole mount immunostained for the peripheral macrophage marker Ccr2 (red) and Iba1 (white). The amoeboid shaped Iba1+ cells are positive for Ccr2 (arrows) while branched Iba1+ cells are negative. (C) Single z-plane (0.8 μm) from a Cx3cr1^gfp/gfp DBA/2J retina immunostained for the peripheral macrophage marker Ccr2 (red) and Iba1 (white). As in (B) the amoeboid shaped Iba1+ cells are positive for Ccr2 (arrows) while branched Iba1+ cells are negative. (D) There is significantly more expression of Ccr2 and Ccl2 in Cx3cr1^gfp/gfp DBA/2J whole retina cDNA (3 and 2-fold more expression respectively, ^*p < 0.05; Student's t-test; error bars represent the SEM, 5 retinas per condition). (E) Gating parameters were set by dissociated retinal cells not exposed to antibodies. 56,935 viable cells collected. (F) Left, representative plot of DBA/2J assessing CD45⁺ CD11b⁺ (gate shown) from 129,000 to 200,000 viable cells. Right, histograms and percent CCR2⁺ of gated CD45⁺CD11b⁺ population. (G) Left, representative plots of Cx3cr1^gfp/gfp DBA/2J assessing CD45⁺ CD11b⁺ (gate shown) from 129,000 to 200,000 viable cells as in F. Right, histograms and percent CCR2⁺ of gated CD45⁺CD11b⁺ population. (H) Top, total number and percent of CCR2⁺ and CCR2⁻ of all CD45⁺CD11b⁺ cells analyzed by flow cytometry in DBA/2J n = 8 pooled retinas (293,780 cells collected). Bottom, total number and percent of CCR2⁺ and CCR2⁻ of all CD45⁺CD11b⁺ cells analyzed by flow cytometry in Cx3cr1^gfp/gfp DBA/2J n = 6 pooled retinas (453,727 cells collected). (I): There is ~3-fold more expression of Nos2 but 33% less expression of IL1β in FACS sorted myeloid cells (n = 8 DBA/2J pooled retinas and n = 6 CX3CR1^gfp/gfp DBA/2J pooled retinas). Scale bars: 50 μm (B,C) 10 μm (A).