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Detection of Reactive Oxygen Species Using MitoSOX and CellROX in Zebrafish
使用 MitoSOX 和 CellROX 在斑马鱼中检测活性氧类   

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Abstract

The production of free radicals is the result of normal cellular metabolism. Free radicals are involved in innumerable different cellular and biological functions such as signaling, proliferation, cell death, aging, inflammation, etc. Under physiological conditions, the levels of reactive oxygen species (ROS) are strictly regulated by the cells. However, during stressful conditions such as oxidative stress, ROS levels increase causing damages to different molecules like DNA, lipids and proteins. Increased levels of ROS have been associated with a growing list of different diseases. In this protocol, we used MitoSOX and CellROX Green oxidative stress probes to label the intracellular ROS and detect the fluorescence using cell sorting and confocal analyses.

Keywords: Zebrafish(斑马鱼), Reactive Oxygen Species(活性氧), Oxidative stress(氧化应激)

Materials and Reagents

  1. 10 cm sterile Petri dishes (Thermo Fisher Scientific, Fisher Scientific, catalog number: FB0875711Z )
  2. 1.5 ml tubes (Eppendorf, catalog number: 022363204 )
  3. 35 mm TC-Treated culture dish (Corning, catalog number: 430165 )
  4. Falcon round-bottom polystyrene 5 ml tubes (Corning, Falcon®, catalog number: 352003 )
  5. Sterile 40 μm cell strainer (Corning, catalog number: 431750 )
  6. 35 mm glass bottom dish (Cellvis, catalog number: D35-20-1-N )
  7. 3 ml transfer pipette (Sigma-Aldrich, catalog number: Z135003 )
  8. Zebrafish adults (wild-type or mutant strains)
  9. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  10. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  11. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
  12. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 63138 )
  13. ddH2O (Sigma-Aldrich, catalog number: W4502 )
  14. DMEM/F-12 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11330-032 )
  15. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 16000-036 )
  16. Tricaine (Sigma-Aldrich, catalog number: A5040 )
  17. 10x trypsin-EDTA (0.5%), no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 15400-054 )
  18. Tris-EDTA buffer solution (pH 9.0) (Sigma-Aldrich, catalog number: SRE0063 )
  19. Collagenase P (Sigma-Aldrich, catalog number: COLLP-RO )
  20. Phosphate buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, GibcoTM, catalog number: 10010-023 )
  21. Hank's balanced salt solution (HBSS), with calcium and magnesium, no phenol red (HBSS/Ca/Mg) (Thermo Fisher Scientific, GibcoTM, catalog number: 14025-092 )
  22. MitoSOXTM red mitochondrial superoxide indicator (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: M36008 )
  23. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
  24. 7-aminoactinomycin D (7-AAD) (BD, BD PharmingenTM, catalog number: 559925 )
  25. Annexin V-APC (BD, BD PharmingenTM, catalog number: 550474 )
  26. Annexin V binding buffer, 10x concentrate (BD, BD PharmingenTM, catalog number: 556454 )
  27. CellROX® green reagents, for oxidative stress detection (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C10444 )
  28. CellROX® green flow cytometry assay kit (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C10492 )
  29. CertifiedTM low-melting agarose (Bio-Rad Laboratories, catalog number: 1613111 )
  30. Instant Ocean aquarium sea salt mixture (Spectrum Brand, Instant Ocean, catalog number: SS15-10 )
  31. 24x tricaine solution (see Recipes)
  32. DMEM/F-12 medium (see Recipes)
  33. 60x E3 embryo medium stock solution (see Recipes)
  34. Homogenization medium (see Recipes)
  35. 5 mM MitoSOX stock solution (see Recipes)

Equipment

  1. Dissecting forceps - microdissection (DR Instruments, catalog number: 18 )
  2. Ultrafine single deer hair with handle (Ted Pella, catalog number: 119 )
  3. Thermoblock (Sigma-Aldrich, catalog number: Z605271 )
  4. Manual pipette PR-200 (Mettler-Toledo, Rainin, catalog number: 17008652 )
  5. Standard tabletop centrifuges (Eppendorf, catalog number: 022620401 )
  6. Dissecting microscope (Leica, model: Leica S8 APO )
  7. FACS-Fluorescence-activated cell sorter [e.g., FACSAria II equipped with FACSDiva software using the blue 488-nm and red 640-nm lasers (BD)]
  8. Confocal microscope [e.g., LSM 510 NLO Meta system mounted on an Axiovert 200M microscope (Carl Zeiss) with a Plan-Apochromat 10x/0.45 or a Plan-Apochromat 20x/0.75 objective lens]

Software

  1. FACSDiva software

Procedure

  1. Single cell homogenization and ROS (MitoSOX and CellROX Green) staining
    1. Grow zebrafish embryos at 28 °C in ~25 ml of fish water or 1x E3 embryo medium until the desired stage.
    2. Pre-heat the homogenization solution and the DMEM-10% FBS at 28 °C.
    3. Collect the embryos and manually dechorionate them with a pair of forceps with sharp tips after ~24 h post fertilization (hpf), if necessary. Use one forceps to hold the external membrane (chorion), then make a tear using the other forceps and gently remove the chorion.
    4. Transfer dechorionated embryos to 1.5 ml tubes using a plastic pipette.
      Note: The quality of the homogenization will depend on the stage and the number of embryos used. Do not exceed the number of 50-60 embryos at 5 days post fertilization (dpf) embryos/tube.
    5. Remove most of the water or 1x E3 embryo medium without damaging or drying the embryos.
    6. Euthanize embryos with ~400 μl of 24x tricaine stock solution and leave on ice for 5 min.
    7. Wash twice with 1 ml of 1x PBS for 1-2 min.
    8. Add 600 μl of homogenization solution (collagenase P + trypsin-EDTA in 1x PBS) and incubate at 28 °C in a thermoblock for 20-30 min depending on the developmental stage or until the complete homogenization of each sample.
    9. During the incubation time, periodically (~every 1-2 min) homogenate the samples pipetting up and down with a 200 μl pipette. During the incubation time a progressive homogenization of the samples should be noted.
    10. Add 700 μl of DMEM-10% FBS, mix by pipetting or vortexing and then centrifuge the cells for 2 min at 600 x g at room temperature (RT).
    11. Discard supernatant and resuspend the cells in 1 ml 1x PBS.
    12. Centrifuge the cells for 2 min at 600 x g.
    13. Resuspend the cells in 1 ml of DMEM-10% FBS.
    14. Keep the cells at 28 °C in DMEM-10% FBS until the staining in order to increase the survival of the cells.
      Note: Alternatively, the cells can be stored at 4 °C in order to minimize the level of oxidative stress induced by cell homogenization. However, we noticed an increased level of apoptotic and dead cells with the incubation at 4 °C.
    15. Aliquots the cells in different tubes and stain the cells with the different staining solutions at 28 °C in a thermoblock in the dark.

  2. Oxidative stress (MitoSOX and CellROX Green), apoptosis and cell death staining
    1. Centrifuge the cells for 2 min at 600 x g at RT.
    2. Remove supernatant and resuspend the cells in prewarmed HBSS/Ca/Mg at 28 °C.
    3. Centrifuge the cells, remove the supernatant and stain the cells with 400 μl of 2.5-5 μM solution of MitoSOX in HBSS/Ca/Mg for 15 min or a 1-2.5 μM solution of CellROX Green in DMEM-10% FBS for 30 min at 28 °C, in the dark. Wash the cells three times with 1 ml of prewarmed HBSS/Ca/Mg to remove the excess of the probes.
    4. To test the viability of the cells after homogenization, an Annexin V-APC and 7-AAD double staining can be performed in parallel for the oxidative stress staining. For Annexin V-APC and 7-AAD double staining cells were centrifuged, washed with 500 μl of prewarmed 1x binding buffer at 28 °C (from Annexin V-APC kit) and then incubated in 600 μl of 1x binding buffer with 3 μl of the probes for 15 min at 28 °C in the dark. After the staining, wash the cells three times with 500 μl of prewarmed 1x binding buffer.
    5. Filter the cell solution through a sterile 40 μm nylon mesh before to FACS analysis.
    6. Transfer the cells to a 5 ml tube and then sort the cells with a FACS.
    7. Flow cytometry data were collected on a FACSAria II equipped with FACSDiva software using the blue 488-nm laser for 7-AAD (550/647 nm, Excitation/Emission peaks), CellROX green (CellROX green flow cytometry assay kit: 508/525 nm; CellROX green reagent: 485/520 nm) and MitoSOX (510/580 nm) reagents and the red 640-nm laser (BD) for APC signal (650/660 nm) (see Figure 2 for representative analysis).

  3. Whole mount staining of ROS (MitoSOX and CellROX Green)
    1. Grow zebrafish embryos at 28 °C in ~25 ml of fish water or 1x E3 embryo medium until the desired stage.
    2. Collect the embryos and manually dechorionate them with forceps, if necessary.
    3. Transfer the embryos in a 1.5 ml tube (10-15 embryos/tube).
    4. Prepare low-melting agarose solution using fish water and a specific amount of low-melting agarose powder according to the desired percentage of the gel using a microwave. Let it cool down for 5 min at RT then prepare some 1 ml aliquots in 1.5 ml tubes and keep them at ~37 °C in the thermoblock ready to be used (the percentage of agarose depends on the stage of the embryos: usually 0.5-0.8% until 1-2 dpf, ~1% after 2 dpf).
    5. Remove fish water or 1x E3 embryo medium and wash the embryos once in 1 ml of HBSS/Ca/Mg.
    6. Remove the HBSS/Ca/Mg without damaging or drying the embryos and add the specific staining solution as previously indicated (see step B3).
    7. Incubate at 28 °C in the dark. Usually 10-30 min represents a good starting point, but the length of the incubation should be optimized based on the developmental stage, the staining solution and the oxidative status of the sample.
    8. After the incubation time wash three times with 1 ml of prewarmed HBSS/Ca/Mg at 28 °C, then anesthetize the embryos in 1ml of 1x tricaine in 1x E3 embryo medium for ~5 min.
    9. Transfer the embryos individually in a 35 mm glass bottom dish, remove the excess of liquid (without damaging or drying the embryos) and mount them in ~150 μl/embryo of low melting agarose gel. Orientate each embryo as desired using a small paintbrush under a dissecting microscope. After gel solidification, gently cover the embryos with enough volume of 1x tricaine in 1x E3 embryo medium in order to anesthetize them (see Figure 1).
    10. Analyze the embryos using a confocal microscope and a 488 nm laser. The MitoSOX and CellROX Green wavelengths of excitation and emission are: Mitosox 510/580 nm, CellROX green flow citometry assay kit: 508/525 nm and CellROX green reagent: 485/520 nm (see Figure 2D).
    11. After the imaging, the embryos can be recovered from the agarose using a pair of forceps in order to be genotyped, if necessary.


      Figure 1. Mounting of live embryos in low melting agarose for confocal analysis. A. Tools required for zebrafish embryos inclusions in agarose gel. B. Prepare a small Petri dish with 1x tricaine in 1x E3 embryo medium in order to anesthetize the embryo (Petri dish on the left) and a 35 mm glass bottom dish (Petri dish on the right). Transfer 3 or more embryos in the anesthetic solution and wait until they are anesthetized. C. Transfer the anesthetized embryos to the 35 mm glass bottom dish and pour gently the agarose solution using a PR-200 pipette. D. Gently orientate the embryos using the small paintbrush without damaging them and until the agarose gel is solid enough to block them in the desired position. E. At the end of the solidification, the gel should appear slightly opaque and dense if touched using the paintbrush. In the specific case, 5 dpf embryos have been orientated in a lateral position. Slowly fill the Petri dish with 1x tricaine in 1x E3 embryo medium in order to avoid the detachment of the gel from the glass.

Representative data



Figure 2. Representative analysis of oxidative stress in WT and mutant embryos using MitoSOX and CellROX staining. A-C. Representative quantitative analysis by flow cytometry of oxidative stress using MitoSOX Red (A and B) or CellROX Green (C) on 4 and 5 dpf mutant and control embryos. Error bars indicate standard deviation; **P < 0.01; ***P < 0.001 using an unpaired Student’s t-test. D. Whole mount staining with the MitoSOX or CellROX probes followed by confocal microscopy in the tail region of WT and mutant embryos at the indicated stages. As internal positive control, embryos at the stages of 4 or 5 dpf were subjected to tail resection 30 min before being stained with MitoSOX or CellROX solutions. If tail resection cannot be performed, because of the stage analyzed or because it could interfere with the specific conditions of the experiment or the fish line used, an alternative positive control could be represented by the treatment of the embryos as indicated in Mugoni et al. (2014). Yellow arrowheads in the insets indicate MitoSOX- or CellROX-positive cells in CHT regions. CHT: Caudal Hematopoietic Tissue (from Rissone et al., 2015).

Recipes

  1. 24x tricaine stock solution
    400 mg tricaine powder in 97.9 ml ddH2O
    Use 2.1 ml 1 M Tris (pH 9) to adjust pH to 7.
    Store the solution in refrigerator for short period (1-2 weeks) or in the freezer in aliquots (1 month).
    Dilute to 1x with 1x E3 embryo medium (or fish water) to anesthetize the embryos.
  2. DMEM/F-12 medium
    Supplemented with 10% fetal bovine serum (FBS)
  3. 60x E3 embryo medium stock solution
    17.2 g NaCl
    0.76 g KCl
    2.9 g CaCl2·2H2O
    4.9 g MgSO4·7H2O
    Dissolve in 1 L ddH2O
  4. Homogenization solution
    10 mg/ml collagenase P
    0.05% trypsin-EDTA
    1x PBS
  5. 5 mM MitoSOX stock solution
    Dissolve the contents (50 μg) of one vial of MitoSOX mitochondrial superoxide indicator in 13 μl of DMSO.

Acknowledgments

This protocol was modified from Behra et al. (2012) and Mugoni et al. (2014), and developed in the Dr. Fabio Candotti Lab. Disorders of Immunity Section, Genetics and Molecular Biology Branch, NHGRI, NIH. This protocol was supported by funding from the Intramural Research Program of the National Human Genome Research Institute (to F. Candotti, R. Sood, and S. Chandrasekharappa).

References

  1. Behra, M., Gallardo, V. E., Bradsher, J., Torrado, A., Elkahloun, A., Idol, J., Sheehy, J., Zonies, S., Xu, L., Shaw, K. M., Satou, C., Higashijima, S., Weinstein, B. M. and Burgess, S. M. (2012). Transcriptional signature of accessory cells in the lateral line, using the Tnk1bp1:EGFP transgenic zebrafish line. BMC Dev Biol 12: 6.
  2. Mugoni, V., Camporeale, A. and Santoro, M. M. (2014). Analysis of oxidative stress in zebrafish embryos. J Vis Exp(89).
  3. Rissone, A., Weinacht, K. G., la Marca, G., Bishop, K., Giocaliere, E., Jagadeesh, J., Felgentreff, K., Dobbs, K., Al-Herz, W., Jones, M., Chandrasekharappa, S., Kirby, M., Wincovitch, S., Simon, K. L., Itan, Y., DeVine, A., Schlaeger, T., Schambach, A., Sood, R., Notarangelo, L. D. and Candotti, F. (2015). Reticular dysgenesis-associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress. J Exp Med 212(8): 1185-1202.

简介

自由基的产生是正常细胞代谢的结果。 自由基涉及无数不同的细胞和生物学功能,例如信号传导,增殖,细胞死亡,衰老,炎症等。 在生理条件下,活性氧(ROS)的水平被细胞严格调节。 然而,在应激条件例如氧化应激期间,ROS水平增加导致对不同分子如DNA,脂质和蛋白质的损伤。 增加的ROS水平与不断增加的不同疾病列表相关联。 在这个协议中,我们使用MitoSOX和CellROX绿色氧化应激探针标记细胞内ROS和使用细胞分选和共聚焦分析检测荧光。

关键字:斑马鱼, 活性氧, 氧化应激

材料和试剂

  1. 10cm无菌培养皿(Thermo Fisher Scientific,Fisher Scientific,目录号:FB0875711Z)
  2. 1.5ml管(Eppendorf,目录号:022363204)
  3. 35mm TC处理的培养皿(Corning,目录号:430165)
  4. Falcon圆底聚苯乙烯5ml管(Corning,Falcon ,目录号:352003)
  5. 无菌40μm细胞过滤器(Corning,目录号:431750)
  6. 35mm玻璃底皿(Cellvis,目录号:D35-20-1-N)
  7. 3ml转移移液管(Sigma-Aldrich,目录号:Z135003)
  8. 斑马鱼成虫(野生型或突变株)
  9. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  10. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  11. 氯化钙二水合物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C5080)
  12. 硫酸镁七水合物(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:63138)
  13. ddH 2 O(Sigma-Aldrich,目录号:W4502)
  14. DMEM/F-12培养基(Thermo Fisher Scientific,Gibco TM ,目录号:11330-032)
  15. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:16000-036)
  16. Tricaine(Sigma-Aldrich,目录号:A5040)
  17. 10x胰蛋白酶-EDTA(0.5%),无酚红(Thermo Fisher Scientific,Gibco TM,目录号:15400-054)
  18. Tris-EDTA缓冲液(pH9.0)(Sigma-Aldrich,目录号:SRE0063)
  19. 胶原酶P(Sigma-Aldrich,目录号:COLLP-RO)
  20. 磷酸盐缓冲盐水(PBS),pH 7.4(Thermo Fisher Scientific,Gibco TM ,目录号:10010-023)
  21. 具有钙和镁,没有酚红(HBSS/Ca/Mg)的Hank's平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM,目录号:14025-092)
  22. MitoSOX TM红色线粒体超氧化物指示剂(Thermo Fisher Scientific,Molecular Probes TM ,目录号:M36008)
  23. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2650)
  24. 7-氨基放线菌素D(7-AAD)(BD,BD Pharmingen ,目录号:559925)
  25. 膜联蛋白V-APC(BD,BD Pharmingen TM ,目录号:550474)
  26. 膜联蛋白V结合缓冲液,10倍浓缩物(BD,BD Pharmingen ,目录号:556454)
  27. 用于氧化应激检测的CellROX?绿色试剂(Thermo Fisher Scientific,Molecular Probes TM ,目录号:C10444)
  28. CellROX?green flow cytometry assay kit(Thermo Fisher Scientific,Molecular Probes TM ,目录号:C10492)
  29. 认证的低熔点琼脂糖(Bio-Rad Laboratories,目录号:1613111)
  30. 即时海洋水族馆海盐混合物(Spectrum Brand,Instant Ocean,目录号:SS15-10)
  31. 24x三卡因溶液(见配方)
  32. DMEM/F-12培养基(见配方)
  33. 60x E3胚胎培养基原液(见配方)
  34. 均质介质(见配方)
  35. 5 mM MitoSOX储备溶液(见配方)

设备

  1. 解剖钳 - 显微切割(DR Instruments,目录号:18)
  2. 超细单头发手柄(Ted Pella,目录号:119)
  3. Thermoblock(Sigma-Aldrich,目录号:Z605271)
  4. 手动移液器PR-200(Mettler-Toledo,Rainin,目录号:17008652)
  5. 标准台式离心机(Eppendorf,目录号:022620401)
  6. 解剖显微镜(Leica,型号:Leica S8 APO)
  7. FACS-荧光激活的细胞分选仪[例如,FACSAria II,装备有使用蓝色488-nm和红色640-nm激光(BD)的FACSDiva软件]。
  8. 例如,安装在具有Plan-Apochromat 10x/0.45或Plan-Apochromat 20x/0.75物镜的Axiovert 200M显微镜(Carl Zeiss)上的LSM 510NLO Meta系统[例如 >

软件

  1. FACSDiva软件

程序

  1. 单细胞匀浆和ROS(MitoSOX和CellROX Green)染色
    1. 在28℃下在〜25ml鱼水或1x E3胚胎培养基中生长斑马鱼胚胎,直到期望的阶段。
    2. 在28℃下预热匀浆溶液和DMEM-10%FBS
    3. 收集胚胎,并手动dechorionate他们与一对镊子尖锐尖后24小时后受精(hpf),如果必要。使用一个钳子夹住外膜(绒毛膜),然后使用其他镊子撕裂,轻轻除去绒毛膜。
    4. 使用塑料移液管转移dechorionated胚胎到1.5毫升管。
      注意:均质化的质量将取决于所使用的胚胎的阶段和数量。在受精(dpf)胚胎/管后5天,不要超过50-60个胚胎。
    5. 去除大部分的水或1x E3胚胎培养基,而不损坏或干燥胚胎
    6. 安慰死胚胎与〜400微升24x三卡因储备液,并在冰上留下5分钟
    7. 用1ml 1×PBS洗涤两次,每次1-2分钟
    8. 加入600μl匀浆溶液(胶原酶P +胰蛋白酶-EDTA在1×PBS中),并在28℃在加热块中孵育20-30分钟,取决于发育阶段或直到每个样品完全匀浆。
    9. 在孵育时间期间,定期(〜每1-2分钟)匀化用200μl移液管向上和向下移液的样品。在孵育期间,应注意样品的渐进均匀化
    10. 加入700μlDMEM-10%FBS,通过吸移或涡旋混合,然后在室温(RT)下以600×g离心细胞2分钟。
    11. 弃去上清液并将细胞重悬在1ml 1×PBS中
    12. 以600×g离心细胞2分钟。
    13. 将细胞重悬在1ml DMEM-10%FBS中
    14. 保持细胞在28℃在DMEM-10%FBS直到染色,以增加细胞的存活。
      注意:或者,可以在4℃下储存细胞,以使由细胞匀浆诱导的氧化应激的水平最小化。然而,我们注意到在4℃孵育时凋亡和死亡细胞的水平增加。
    15. 将细胞在不同的管中等分,并在28℃下在黑暗的加热块中用不同的染色溶液染色细胞。

  2. 氧化应激(MitoSOX和CellROX Green),凋亡和细胞死亡染色
    1. 在室温下以600×g离心细胞2分钟。
    2. 取出上清液,并在28℃预热的HBSS/Ca/Mg中重悬细胞
    3. 离心细胞,去除上清液,并用400μl的2.5-5μMMitoSOX的HBSS/Ca/Mg溶液15分钟或1-2.5μM的CellROX Green的DMEM-10%FBS溶液染色细胞30分钟在28℃,在黑暗中。用1ml预热的HBSS/Ca/Mg洗涤细胞三次,以除去过量的探针。
    4. 为了测试匀浆后细胞的活力,可以平行进行膜联蛋白V-APC和7-AAD双染色用于氧化应激染色。对于膜联蛋白V-APC和7-AAD双染色,将细胞离心,用500μl预热的1×结合缓冲液在28℃(来自Annexin V-APC试剂盒)洗涤,然后在600μl1x结合缓冲液中与3μl探针在28℃在黑暗中15分钟。染色后,用500μl预热的1x结合缓冲液洗涤细胞三次。
    5. 在进行FACS分析之前,通过无菌的40μm尼龙网过滤细胞溶液
    6. 将细胞转移到5ml管中,然后用FACS分选细胞
    7. 在装备有FACSDiva软件的FACSAria II上,使用用于7-AAD的蓝色488-nm激光(550/647nm,激发/发射峰),CellROX green(CellROX green flow cytometry assay kit:508/; CellROX绿色试剂:485/520nm)和MitoSOX(510/580nm)试剂和红色640nm激光(BD)用于APC信号(650/660nm)(参见图2的代表性分析) >
  3. ROS的整体染色(MitoSOX和CellROX Green)
    1. 在28℃下在〜25ml鱼水或1x E3胚胎培养基中生长斑马鱼胚胎,直到期望的阶段。
    2. 收集胚胎,并手动deufforionate他们用镊子,如果必要
    3. 转移胚胎在1.5毫升管(10-15胚胎/管)
    4. 根据所需的凝胶百分比,使用微波,使用鱼水和特定量的低熔点琼脂糖粉末制备低熔点琼脂糖溶液。让它在室温下冷却5分钟,然后在1.5ml试管中制备一些1ml等分试样,并保持在〜37℃,准备使用的热块中(琼脂糖的百分比取决于胚胎的阶段:通常0.5- 0.8%直至1-2dpf,〜2dpf后〜1%)
    5. 取出鱼水或1x E3胚胎培养基,并在1ml HBSS/Ca/Mg中清洗胚胎一次。
    6. 取出HBSS/Ca/Mg,而不损坏或干燥胚胎,并添加如前所述的特定染色溶液(参见步骤B3)。
    7. 在28℃在黑暗中孵育。通常10-30分钟代表良好的起点,但孵育的长度应根据发育阶段,染色溶液和样品的氧化状态进行优化。
    8. 培养时间后,用1ml预热的HBSS/Ca/Mg在28℃洗涤3次,然后在1×1X三卡因的1x E3胚胎培养基中麻醉胚胎〜5分钟。
    9. 转移胚胎单独在一个35毫米玻璃底盘,删除过量的液体(不伤害或干燥胚胎),并将其安装在〜150微升/低熔点琼脂糖凝胶胚胎。根据需要使用小画笔在解剖显微镜下定位每个胚胎。凝胶凝固后,轻轻覆盖胚胎与1×E3胚胎培养基中的足够体积的一个三卡因,以麻醉他们(见图1)。
    10. 使用共聚焦显微镜和488 nm激光分析胚胎。 MitoSOX和CellROX Green的激发和发射波长是:Mitosox 510/580 nm,CellROX绿色流动检测试剂盒:508/525 nm和CellROX绿色试剂:485/520 nm(参见图2D)。
    11. 成像后,如果需要,可以使用一对镊子从琼脂糖中回收胚胎以进行基因分型。


      图1.将活胚置于低熔点琼脂糖中进行共聚焦分析。A.琼脂糖凝胶中斑马鱼胚胎内含物所需的工具。 B.在1x E3胚胎培养基中制备具有1x三卡因的小培养皿,以便麻醉胚胎(左侧的培养皿)和35mm玻璃底培养皿(右侧的培养皿)。转移3个或更多的胚胎在麻醉溶液,并等待,直到他们麻醉。 C.将麻醉的胚胎转移到35mm玻璃底盘,并使用PR-200移液管轻轻地倾倒琼脂糖溶液。 D.使用小画笔轻轻地定向胚胎,而不损坏它们,直到琼脂糖凝胶足够坚固以阻止它们在期望的位置。 E.在凝固结束时,如果使用画笔触摸,凝胶应显得稍微不透明和致密。在具体情况下,5个dpf胚胎已经定向在侧向位置。在1x E3胚胎培养基中缓慢地填充1克三卡因,以避免凝胶从玻璃上剥离。

代表数据



图2.使用MitoSOX和CellROX染色的WT和突变体胚胎中氧化应激的代表性分析。 A-C。使用MitoSOX红色(A和B)或CellROX绿色(C)在4和5 dpf突变体和对照胚胎上的氧化应激的流式细胞术的代表性定量分析。误差线表示标准偏差; ** P 0.01; *** P 0.001使用不成对的Student's t 测试。 D.用MitoSOX或CellROX探针进行整体染色,然后在指定阶段的WT和突变体胚胎的尾部区域进行共聚焦显微镜检查。作为内部阳性对照,在用MitoSOX或CellROX溶液染色之前,对4或5dpf阶段的胚胎进行尾部切除30分钟。如果不能进行尾部切除,由于分析的阶段,或者因为它可能干扰实验的特定条件或使用的鱼线,可以通过如Mugoni等人所述的胚胎处理来代表备选的阳性对照, et al 。 (2014)。插图中的黄色箭头表示CHT区域中的MitoSOX-或CellROX-阳性细胞。 CHT:尾侧造血组织(来自Rissone等人,2015)。

食谱

  1. 24x三卡因储备液
    400mg三卡因粉末在97.9ml ddH 2 O中的溶液 使用2.1ml 1M Tris(pH 9)将pH调节至7.
    将溶液在冰箱中短时间(1-2周)或在冰箱中等分(1个月)。
    用1x E3胚胎培养基(或鱼水)稀释至1倍以麻醉胚胎
  2. DMEM/F-12培养基
    补充10%胎牛血清(FBS)
  3. 60x E3胚胎培养基原液
    17.2克NaCl 0.76克KCl
    2.9g CaCl 2·2H 2 O·h/v 4.9g MgSO 4·7H 2 O·h/v 溶解于1L ddH 2 O中
  4. 均质溶液
    10mg/ml胶原酶P
    0.05%胰蛋白酶-EDTA
    1x PBS
  5. 5 mM MitoSOX储备液
    将一个小瓶的MitoSOX线粒体过氧化物指示剂的内容物(50μg)溶解在13μl的DMSO中。

致谢

该协议是从Behra等人修改的 。 (2012)和Mugoni等人。 (2014),并在Fabio Candotti实验室博士开发。免疫部分,遗传和分子生​​物学分支,NHGRI,NIH。这个协议得到了国家人类基因组研究所的校内研究计划(F. Candotti,R.Sood和S.Chandrasekharappa)的资助。

参考文献

  1. Behra,M.,Gallardo,VE,Bradsher,J.,Torrado,A.,Elkahloun,A.,Idol,J.,Sheehy,J.,Zonies,S.,Xu,L.,Shaw,KM, C.,Higashijima,S.,Weinstein,BM和Burgess,SM(2012)。 
  2. Mugoni,V.,Camporeale,A.和Santoro,MM(2014)。  斑马鱼胚胎中氧化应激的分析。

    (89)。
  3. Rissone,A.,Weinacht,KG,la Marca,G.,Bishop,K.,Giocaliere,E.,Jagadeesh,J.,Felgentreff,K.,Dobbs,K.,Al-Herz,W.,Jones,M 。,Chandrasekharappa,S.,Kirby,M.,Wincovitch,S.,Simon,KL,Itan,Y.,DeVine,A.,Schlaeger,T.,Schambach,A.,Sood,R.,Notarangelo, Candotti,F。(2015)。  网状发育不全相关AK2保护造血干细胞和祖细胞发育免受氧化应激。 212(8):1185-1202。
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引用:Rissone, A. and Candotti, F. (2016). Detection of Reactive Oxygen Species Using MitoSOX and CellROX in Zebrafish. Bio-protocol 6(19): e1941. DOI: 10.21769/BioProtoc.1941.
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