Fig. 3: Identification of mNPPFC-labeled neutrophils in the infarcted heart.
From: Non-invasive mapping of systemic neutrophil dynamics upon cardiovascular injury
a, For identification of mNPPFC-labeled neutrophils in the infarcted heart, mice received injections of rhodamine-tagged mNPPFCs or ConPFCs 2 h before induction of MI and hearts were excised 2 h after induction of MI. The infarct area was visualized by 2,3,5-triphenyltetrazolium chloride (TTC) staining, which was found to colocalize with strong rhodamine signals derived from mNPPFC-labeled cells (bottom). By contrast, ConPFCs led to much weaker and spread-out signals only (top). Magnifications and histograms (fourth and fifth columns) demonstrated distinct signals for mNPPFCs but only diffuse patterns for ConPFCs. b, Left, examination of infarct (MI) and remote (RM) regions revealed strong rhodamine labeling colocalized with Ly6G staining in the injured tissue. Right, quantification confirmed the selective uptake of mNPPFCs versus ConPFCs. c, Analysis of mNPPFC uptake by cardiac neutrophils (left, Ly6G staining), macrophages (middle, major histocompatibility complex (MHC) II staining) and T cells (right, CD3 staining). Rhodamine signals were colocalized with neutrophils, while macrophages and T cells showed little or no signals. d, Quantification of mean fluorescence intensities (MFI) of individual cell types demonstrated significantly stronger labeling of neutrophils than of macrophages and T cells. e, To further corroborate the histological data, immune cells were isolated from the infarcted heart by Langendorff digestion and analyzed by flow cytometry. To identify the different immune cell clusters, cells were stained for CD45, CD11b, Ly6C and Ly6G. Neutrophils (CD45+CD11b+Ly6C−Ly6G+) were characterized by strong labeling after mNPPFC injection, while classical monocytes (CD45+CD11b+Ly6C+Ly6G−), macrophages (CD45+CD11b+Ly6C−Ly6G−) and lymphocytes (CD45+CD11b−Ly6C−Ly6G−) exhibited only low signal intensities in relation to animals injected with ConPFC. Data are mean ± s.d. of n = 3 (a), n = 3 (b), n = 3 (c), n = 8–48 (d) and n = 6 (e) independent experiments (a–c,e) or individual cell MFI measurements from three independent experiments (d). **P < 0.01, ***P < 0.001, verified by two-sided Student’s t-test (b) or one-way ANOVA.
