氯膦酸二鈉脂質(zhì)體清除斑馬魚巨噬細Protocol解決方案

背景介紹：

在生理學的廣泛背景下，進行體內(nèi)巨噬細胞特異性去除的能力仍然是一種有效的手段，可以揭示巨噬細胞的功能。與小鼠模型相比，斑馬魚提供了更方便的成像能力，因為它們從單細胞階段到整個幼蟲發(fā)育過程中都具有光學透明性。這些特性對于進行體內(nèi)細胞特異性去除變得非常重要，以便能夠?qū)崟r跟蹤和驗證目標細胞的消除。斑馬魚中去除巨噬細胞的方法有多種，包括基因去除（例如irf8敲除）、化學遺傳學（例如硝基還原酶/甲硝唑系統(tǒng)）和基于毒素的去除（例如使用荷蘭Liposoma的氯膦酸鹽二鈉脂質(zhì)體Clodronateliposomes）。使用氯膦酸鹽二鈉脂質(zhì)體Clodronateliposomes來誘導吞噬脂質(zhì)體后巨噬細胞凋亡在去除巨噬細胞以及測試它們的吞噬能力方面都是有效的。在這里，我們描述了一種通過靜脈注射含熒光右旋糖酐的氯膦酸鹽二鈉脂質(zhì)體Clodronateliposomes系統(tǒng)性去除斑馬魚幼蟲巨噬細胞的詳細方案。與熒光右旋糖酐的共同注射允許實時跟蹤巨噬細胞的去除，從驗證成功的靜脈注射到巨噬細胞對分子攝取及其最終死亡。為了驗證巨噬細胞高程度的去除，可以通過在早期幼蟲階段進行氯膦酸鹽二鈉脂質(zhì)體Clodronateliposomes注射時快速的中性紅活染料染色來確定腦部巨噬細胞（小膠質(zhì)細胞）的消除程度。

Background：

The ability to conduct in vivo macrophage-specific depletion remains an effective means to uncover functions of macrophages in a wide range of physiological contexts. Compared to the murine model, zebrafish offer superior imaging capabilities due to their optical transparency starting from a single-cell stage to throughout larval development. These qualities become important for in vivo cell specific depletions so that the elimination of the targeted cells can be tracked and validated in real time through microscopy. Multiple methods to deplete macrophages in zebrafish are available, including genetic (such as an irf8 knockout), chemogenetic (such as the nitroreductase/metronidazole system), and toxin-based depletions (such as using clodronate liposomes). The use of clodronate-containing liposomes to induce macrophage apoptosis after phagocytosing the liposomes is effective in depleting macrophages as well as testing their ability to phagocytose. Here we describe a detailed protocol for the systemic depletion of macrophages in zebrafish larvae by intravenous injection of liposomal clodronate supplemented with fluorescent dextran conjugates. Co-injection with the fluorescent dextran allows tracking of macrophage depletion in real time starting with verifying the successful intravenous injection to macrophage uptake of molecules and their eventual death. To verify a high degree of macrophage depletion, the level of brain macrophage (microglia) elimination can be determined by a rapid neutral red vital dye staining when clodronate injection is performed at early larval stages.

氯膦酸二鈉脂質(zhì)體清除斑馬魚巨噬細Protocol解決方案：