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LIPOSOMA氯膦酸二钠脂质体清除斑马鱼的单核巨噬细胞

发布时间:2022-11-12   点击次数:272次

Liposomal Clodronate-mediated Macrophage Depletion in the Zebrafish Model
在斑马鱼模型中脂质体氯膦酸盐介导的巨噬细胞耗竭/清除  


Abstract:

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.


摘要:

进行体内巨噬细胞特异性耗竭的能力仍然是在广泛的生理背景下揭示巨噬细胞功能的有效手段。与小鼠模型相比,斑马鱼具有强的成像能力,因为它们从单细胞阶段到整个幼虫发育过程中都具有光学透明度。这些品质对于体内细胞特异性耗竭变得很重要,因此可以通过显微镜实时跟踪和验证目标细胞的消除。有多种方法可以去除斑马鱼中的巨噬细胞,包括遗传(例如 irf8 敲除)、化学遗传(例如硝基还原酶/甲硝唑系统)和基于毒素的耗竭(例如使用氯膦酸盐脂质体)。在吞噬脂质体后使用含氯膦酸盐的脂质体诱导巨噬细胞凋亡可有效消耗巨噬细胞以及测试其吞噬能力。在这里,我们描述了通过静脉注射补充有荧光葡聚糖偶联物的氯膦酸脂质体来全身耗竭斑马鱼幼虫巨噬细胞的详细方案。与荧光葡聚糖共注射可以实时跟踪巨噬细胞耗竭,从验证成功静脉注射到巨噬细胞分子摄取及其最终死亡开始。为了验证巨噬细胞的高度耗竭,当在早期幼虫阶段进行氯膦酸盐注射时,可以通过快速中性红色活体染料染色来确定脑巨噬细胞(小胶质细胞)消除的水平。


Materials and Reagents

1. 1.5 ml microfuge tubes (Eppendorf, SafeLock, catalog number: 0030120086)

2. Polystyrene Petri dish (VWR, catalog number: 25384-342)

3. Thin wall borosilicate glass capillaries, 4 inches, OD 1.5 mm with filament (World Precision Instruments, catalog number: TW150F-4)

4. Glass bottle

5. 7.5 ml transfer pipettes (VWR, catalog number: 414004-005)

6. Low melt agarose (Fisher Scientific, IBI Scientific, catalog number: 50-550-455), store at room temperature

7. PTU (N-Phenylthiourea) (Sigma-Aldrich, catalog number: P7629), store at room temperature; made into PTU solution, store at -20 °C

8. Clodronate Liposomes (Liposoma, catalog number: C-005 ), store at 4-7 °C

9. Control Liposomes (Liposoma, catalog number: P-005 ), store at 4-7 °C

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10. Dextran, Alexa FluorTM 568: 10,000 MW (Invitrogen, catalog number: D22912), store in freezer and protect from light

11. Neutral Red Dye (Sigma-Aldrich, catalog number: N4638), store at room temperature

12. Tricaine (3-amino benzoic acidethylester) (Sigma-Aldrich, catalog number: A-5040) made into tricaine solution, store at -20 °C

13. 50× PTU stock (see Recipes)

14. 25× Tricaine stock solution (100 ml) (see Recipes)

15. 3% Methyl cellulose (see Recipes)

16. 1,000× neutral red solution (see Recipes)

17. 1.5% low melt agarose (see Recipes)


原始文献可以联系靶点科技(北京)有限公司索要。靶点科技专注单核巨噬细胞清除研究多年。有专业技术团队提供免费的技术咨询。氯膦酸二钠脂质体ClodronateLiposomes(LIPOSOMA,CP-005-005)是清除单核巨噬细胞的有力工具,使用其发表的文献频频见于Cell、Nature和Science.

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