Extended Data Fig. 5: Effects of MANF ablation in Cx3cr1pos cells.
From: Aging disrupts MANF-mediated immune modulation during skeletal muscle regeneration
a, Experimental timeline for analysis of animals with conditional ablation of MANF only in Cx3cr1-expressing cells existing prior to muscle injury. b–d, Quantification, by flow cytometry, of myeloid cells (CD11bpos, b), pro-repair macrophages (F4/80posLy6CLow, c), and pro-inflammatory macrophages (LyC6High, d) in 3dpi regenerating QC muscles of ManfCx3cr1WT and ManfCx3cr1Δ mice, treated with tamoxifen prior to the injury (n = 3/condition). e, f, Quantification, by flow cytometry, of macrophages (CD11bposF4/80pos, e) and neutrophils (CD11bposF4/80negLy6Gpos, f) non-injured QC muscles of tamoxifen treated Manffl/fl and ManfCx3cr1Δ mice (n = 4/condition). g, Representative density plots from flow cytometry analysis of myeloid blood cell populations gated on CD11bposF4/80neg population, identifying neutrophils (Ly6Gpos), Ly6Cpos classical monocytes (Ly6GnegLy6Cpos) and Ly6Cneg non-classical monocytes (Ly6GnegLy6CnegCx3cr1pos). h-i, Quantification, by flow cytometry, of myeloid cells (CD11bpos), Ly6Cpos classical monocytes, Ly6Cneg non-classical monocytes and neutrophils in the blood of ManfCx3cr1WT and ManfCx3cr1Δ mice prior to muscle injury (h, n = 6 for ManfCx3cr1WT; n = 4 for ManfCx3cr1Δ), and at 3dpi (i, n = 4/ condition). j, l, Representative density plots from flow cytometry analysis of macrophage populations from ManfCx3cr1WT and ManfCx3cr1Δ animals at 3dpi, showing EdU signal (j) and Apopxin-Green signal (l). For both stains the FMO density plot is shown to define the positive population. k, Quantification, by flow cytometry, of EdUpos macrophages as a percentage of pro-repair and pro-inflammatory populations, in ManfCx3cr1WT and ManfCx3cr1Δ animals at 3dpi (n = 3 for ManfCx3cr1WT; n = 4 for ManfCx3cr1Δ). m, Quantification, by flow cytometry, of Apopxinpos macrophages as a percentage of pro-repair the population, in ManfCx3cr1WT and ManfCx3cr1Δ animals at 3dpi (n = 7 for ManfCx3cr1WT; n = 5 for ManfCx3cr1Δ). Data are represented as average ± s.e.m. and each n represents one animal. p values are from two-tailed Student’s t-test. FSC, Forward Scatter; Edu, 5-ethynyl-2’-deoxyuridine; FMO, Fluorescence Minus One Control.
