The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions methods

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    "name": "The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions methods",
    "description": "Evidence-backed execution summary for The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions methods from The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions.",
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      {
        "@type": "HowToStep",
        "position": 1,
        "name": "The injured myocardium sequentially recruits circulating Ly-6C hi and -6C lo monocytes through modulation of chemokin...",
        "text": "Several mechanisms might explain the selective mobilization of Ly-6C hi monocytes during phase I and Ly-6C lo monocytes during phase II. For instance, the injured myocardium may recruit Ly-6C hi cells that convert into Ly-6C lo cells at the onset of phase II. The myocardium may also recruit circulating Ly-6C hi and -6C lo cells sequentially; a biphasic recruitment of monocytes could result from alterations in the relative number of these cells in peripheral blood and/or from preferential recruitment to the target tissue. To address these issues, we sought to determine how circulating Ly-6C hi and -6C lo monocytes contribute to the pool of monocytes/macrophages in injured hearts, and we measured the following two parameters at the onset of phase I and II: (a) the relative abundance of Ly-6C hi and -6C lo monocytes in the blood, and (b) the intrinsic probability (i.e., on a cell-c..."
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        "position": 2,
        "name": "Cells.",
        "text": "Mice were killed 3 h and on days 1-5, 7, and 16 after myocardial infarction ( n = 3-10 mice per time point). Peripheral blood was drawn via cardiac puncture with citrate solution (100 mM Na-citrate, 130 mM glucose, pH 6.5), as anti-coagulant and mononuclear cells were purified by density centrifugation ( ). Total blood leukocyte numbers were determined using acetic acid lysis solution (3% HEMA 3 Solution II, 94% ddH 2 O, and 3% glacial acetic acid). Spleens were removed, triturated in HBSS (Mediatech, Inc.) at 4°C with the end of a 3-ml syringe, and filtered through nylon mesh (BD Biosciences). The cell suspension was centrifuged at 300 g for 10 min at 4°C. Red blood cells were lysed with ACK lysis buffer, and the splenocytes were washed with HBSS and resuspended in HBSS supplemented with 0.2% (wt/vol) BSA and 1% (wt/vol) FCS. Infarct tissue and healthy hearts we..."
      },
      {
        "@type": "HowToStep",
        "position": 3,
        "name": "mAbs and flow cytometry.",
        "text": "The following antibodies were used: anti-CD90-PE, 53-2.1 (BD Biosciences), -B220-PE, RA3-6B2 (BD Biosciences),-CD49b-PE, DX5 (BD Biosciences), -NK1.1-PE, PK136 (BD Biosciences), -Ly-6G-PE, 1A8 (BD Biosciences), -Ly-6G-FITC, 1A8 (BD Biosciences), CD11b-APC, M1/70 (BD Biosciences), -F4/80-biotin, C1:A3-1 (AbD Serotec), -F4/80-FITC, C1:A3-1 (BioLegend), -CD11c-biotin, HL3 (BD Biosciences), -CD11c-FITC, HL3 (BD Biosciences), -I-A b -biotin, AF6-120.1 (BD Biosciences), -I-A b -FITC, AF6-120.1 (BD Biosciences), -Ly-6C-FITC, AL-21 (BD Biosciences), -Ly-6C-biotin, AL-21 (BD Biosciences), TNF-α-FITC, MP6-XT22 (AbD Serotec), anti-CD43-FITC, S7 (BD Biosciences), -CD62L-FITC, MEL-14 (BD Biosciences), -CD68-FITC, FA-11 (AbD Serotec), -CD86-biotin, GL1 (BD Biosciences), -CD115-PE,..."
      },
      {
        "@type": "HowToStep",
        "position": 4,
        "name": "Monocyte subtype tracking.",
        "text": "Ly-6C hi or -6C lo monocytes were sorted from the spleen of C57BL/6 mice on a FACSAria (BD Biosciences) and labeled with 111 In-oxine according to the manufacturer's protocol (GE Healthcare). In brief, cells were washed with HBSS, spun, and resuspended in 111 In-oxine for 15 min at 37°C, pH 6.5-7.5, and washed twice with HBSS. 111 In-oxine is a widely used, FDA-approved agent that is fully biocompatible as it alters neither cell survival nor function at the doses used (,,, ); hence, we considered labeled monocytes to behave similarly to unlabeled monocytes. Approximately 2 × 10 5 Ly-6C hi or -6C lo monocytes incorporating 20 µCi were injected independently i.v. into C57BL/6 on day 0 (phase I) or 4 d (phase II) after MI. The total amount of activity injected into each animal was measured with a radioisotope calibrator (Capintec, Inc.). After 24 h, recipient anim..."
      },
      {
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        "position": 5,
        "name": "Histopathological analysis.",
        "text": "Histopathology for assessment of healing was performed in mice killed 7 d after MI for the following groups: wild-type C57BL/6 mice, wild-type mice depleted with clodronate-loaded liposomes immediately after coronary ligation, wild-type mice depleted with clodronate-loaded liposomes 3 d after MI, and apoE -/- C57BL6 mice. Hearts were excised and rinsed in PBS and embedded in OCT (Sakura Finetek). Serial 6-µm thick sections were used for immunohistochemical staining for neutrophils (NIMP-R14, Abcam), monocytes/macrophages (Mac-3, M3/84; BD PharMingen), myofibroblasts (α-actin, RB-9010-PO; Neomarkers), and endothelial cells forming capillaries (CD31, MEC13.3; BD Biosciences). Reaction was visualized as a three-step staining procedure in combination with biotinylated secondary antibodies (BA4001; Vector Laboratories) and AEC Substrate kit (Vector Laboratories). To a..."
      }
    ],
    "tool": [
      {
        "@type": "HowToTool",
        "name": "The injured myocardium sequentially recruits circulating Ly-6C hi and -6C lo monocytes through modulation of chemokin..."
      },
      {
        "@type": "HowToTool",
        "name": "In vivo relevance of the biphasic response to healing"
      },
      {
        "@type": "HowToTool",
        "name": "mAbs and flow cytometry."
      },
      {
        "@type": "HowToTool",
        "name": "Monocyte subtype tracking."
      },
      {
        "@type": "HowToTool",
        "name": "Histopathological analysis."
      },
      {
        "@type": "HowToTool",
        "name": "Real-time RT-PCR."
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    ],
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        "name": "Cells."
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        "@type": "HowToSupply",
        "name": "mAbs and flow cytometry."
      },
      {
        "@type": "HowToSupply",
        "name": "Histopathological analysis."
      },
      {
        "@type": "HowToSupply",
        "name": "Real-time RT-PCR."
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    "isBasedOn": {
      "@type": "ScholarlyArticle",
      "headline": "The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions",
      "datePublished": "2007",
      "author": [
        {
          "@type": "Person",
          "name": "Matthias Nahrendorf"
        },
        {
          "@type": "Person",
          "name": "Filip K. Swirski"
        },
        {
          "@type": "Person",
          "name": "Elena Aikawa"
        },
        {
          "@type": "Person",
          "name": "Lars Stangenberg"
        },
        {
          "@type": "Person",
          "name": "Thomas Wurdinger"
        },
        {
          "@type": "Person",
          "name": "Jose-Luiz Figueiredo"
        },
        {
          "@type": "Person",
          "name": "Peter Libby"
        },
        {
          "@type": "Person",
          "name": "Ralph Weissleder"
        },
        {
          "@type": "Person",
          "name": "Mikael J. Pittet"
        }
      ],
      "identifier": "10.1084/jem.20070885"
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