Liposomal Clodronate-mediated Macrophage Depletion in the Zebrafish Model
在斑馬魚模型中脂質(zhì)體氯膦酸鹽介導(dǎo)的巨噬細(xì)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：

進(jìn)行體內(nèi)巨噬細(xì)胞特異性耗竭的能力仍然是在廣泛的生理背景下揭示巨噬細(xì)胞功能的有效手段。與小鼠模型相比，斑馬魚具有強(qiáng)的成像能力，因?yàn)樗鼈儚膯渭?xì)胞階段到整個(gè)幼蟲發(fā)育過(guò)程中都具有光學(xué)透明度。這些品質(zhì)對(duì)于體內(nèi)細(xì)胞特異性耗竭變得很重要，因此可以通過(guò)顯微鏡實(shí)時(shí)跟蹤和驗(yàn)證目標(biāo)細(xì)胞的消除。有多種方法可以去除斑馬魚中的巨噬細(xì)胞，包括遺傳（例如 irf8 敲除）、化學(xué)遺傳（例如硝基還原酶/甲硝唑系統(tǒng)）和基于毒素的耗竭（例如使用氯膦酸鹽脂質(zhì)體）。在吞噬脂質(zhì)體后使用含氯膦酸鹽的脂質(zhì)體誘導(dǎo)巨噬細(xì)胞凋亡可有效消耗巨噬細(xì)胞以及測(cè)試其吞噬能力。在這里，我們描述了通過(guò)靜脈注射補(bǔ)充有熒光葡聚糖偶聯(lián)物的氯膦酸脂質(zhì)體來(lái)全身耗竭斑馬魚幼蟲巨噬細(xì)胞的詳細(xì)方案。與熒光葡聚糖共注射可以實(shí)時(shí)跟蹤巨噬細(xì)胞耗竭，從驗(yàn)證成功靜脈注射到巨噬細(xì)胞分子攝取及其最終死亡開始。為了驗(yàn)證巨噬細(xì)胞的高度耗竭，當(dāng)在早期幼蟲階段進(jìn)行氯膦酸鹽注射時(shí)，可以通過(guò)快速中性紅色活體染料染色來(lái)確定腦巨噬細(xì)胞（小膠質(zhì)細(xì)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

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)

原始文獻(xiàn)可以聯(lián)系靶點(diǎn)科技（北京）有限公司索要。靶點(diǎn)科技專注單核巨噬細(xì)胞清除研究多年。有專業(yè)技術(shù)團(tuán)隊(duì)提供免費(fèi)的技術(shù)咨詢。氯膦酸二鈉脂質(zhì)體ClodronateLiposomes(LIPOSOMA,CP-005-005)是清除單核巨噬細(xì)胞的有力工具，使用其發(fā)表的文獻(xiàn)頻頻見于Cell、Nature和Science.