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. 2022 Feb 16;219(5):e20210562. doi: 10.1084/jem.20210562

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© 2022 Mysore et al.

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Figure S1. — Gating strategies, CCR2, and CX3CR1 antibody specificity and the analysis of CCR2 and CX3CR1 on blood monocytes. (A) Representative FACS plot of the gating strategy for the analysis of Ly6C and MHCII in monocyte/macrophage populations in the kidney 14 d after NTN induction. Live, CD45⁺ cells (not depicted) were analyzed under the Singlets⁺Ly6G⁻CD3⁻CD11c⁻NK⁻CD11b⁺ gate. Monocyte/macrophage populations were distinguished by Ly6C and MHCII. FSC, forward scatter; SSC, side scatter. (B) Specificity of monoclonal antibodies against CX3CR1 (1:100 dilution) and CCR2 (1:20 dilution) were confirmed by staining blood samples isolated from Wt, Cx3cr1 KO (Cx3cr1^gfp/gfp), Ccr2 KO (Ccr2^rfp/rfp), and DKO (Ccr2^rfp/rfpCx3cr1^gfp/gfp) mice. A representative FACS plot is shown. CX3CR1 and CCR2 expression assessed by antibodies and GFP and RFP were analyzed under a CD45⁺CD11b⁺ gate. (C) Blood was collected from Wt mice on day 14 after NTN induction and analyzed by flow cytometry. Blood leukocytes were divided based on CD45⁺CD11b⁺ and CD45⁺CD11b⁻ gates and further stained for CX3CR1 and CCR2; monocyte markers Ly6C and CD115; and lineage-negative, CD11c, Ly6G, and CD3. Histogram plots (right) show that all CCR2⁺CX3CR1⁺ DP populations express Ly6C and CD115. None of the DP populations express CD11c, Ly6G, or CD3.