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Visualization of ex vivo Neutrophil Extracellular Traps by Fluorescence Microscopy
荧光显微镜法显示体外中性粒细胞胞外杀菌网   

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Abstract

Neutrophil extracellular traps (NETs) are extracellular DNAs decorated with nuclear and granular proteins such as histones, neutrophil elastase or myeloperoxidase. They exhibit fibrous mesh-like, web-like, or string-like structures. Here, we describe our protocol regarding visualization of ex vivo NETs released from neutrophils activated by lipopolysaccharide (LPS) using fluorescence microscopy.

Materials and Reagents

  1. Whole blood from wild-type C57/BL6 mice (Japan SLC, Inc.)
  2. Whole blood from human volunteers
  3. LPS (Escherichia coli, serotype 0111:B4) (Sigma-Aldrich, catalog number: L4391 )
  4. PolymorphprepTM (Axis Shield PoC AS, catalog number: 1114683 )
  5. RPMI 1640 medium (no phenol red) (Life Technologies, catalog number: 32404-014 )
  6. Fetal bovine serum (Life Technologies, catalog number: 12483-020 )
  7. ACK (Ammonium-chloride-potassium) lysing buffer (Lonza, catalog number: 10-548E )
  8. SYTOX Green (Life Technologies, InvitrogenTM, catalog number: S7020 )

Equipment

  1. Glass Pasteur pipets (Iwaki brand, Asahi Techno Glass Corporation)
  2. 96-well plates (TPP Techno Plastic Products AG)
  3. CO2 Incubator (SANYO)
  4. Fluorescence microscopy (Olympus, model: IX71 )

Procedure

  1. Isolation of human neutrophils
    1. Venous blood (6 ml each) was obtained from healthy human volunteers.
    2.  Neutrophils were isolated by density gradient centrifugation using PolymorphprepTM according to the manufacturer's instructions.
    3.  EDTA anti-coagulated blood (the optimal concentration is 1.5 mg per ml of blood) was layered onto 6 ml Polymorphprep solution and centrifuged at 500 x g for 30 min.
    4. The granulocyte fraction was carefully harvested using a glass Pasteur pipette.
    5. Wash the pellet containing granulocytes with 2 ml phosphate buffered saline (PBS).
    6. Centrifuge at 400 x g for 10 min, and resuspend the pellet in 1 ml PBS.
    7. When present, erythrocytes were lysed with ACK lysing buffer.
    8. 1 ml ACK lysing buffer was added to the pellet with residual erythrocytes.
    9. Incubate at room temperature for 5 min with occasional pipetting.
    10. Wash the pellet containing granulocytes with 2 ml PBS.
    11. Centrifuge at 400 x g for 10 min.
    12. Neutrophils were resuspended in 1 ml of RPMI 1640 without phenol red supplemented with 1% fetal bovine serum.
    13. Final neutrophil concentration was determined by hemacytometer. Approximately 0.5~1.0 x 107/ml of neutrophils will be obtained.
    14. Neutrophil purity was confirmed to be routinely >90%, as assessed by May-Grünwald Giemsa staining on the blood smear.
    15. In brief, immerse the air-dried smear slide in 1 ml May-Grunwald solution for 1 min.
    16. Add an equal part of phosphate buffer and incubate for 3 min.
    17. Pour off the stain and wash the slide with tap water.
    18. Immerse in the 8% Giemsa solution for 20 min.
    19. Wash the slide with tap water and air-dry.

  2. Isolation of murine leukocytes
    1. Heparinized blood was withdrawn from the inferior vena cava of anesthetized wild-type C57/BL6 mice.
    2. In brief, open the abdomen and identify the inferior vena cava between the kidneys.
    3. Use a 25 gauge needle and a 1 ml syringe filled with 50 µl heparin for the prevention of blood coagulation.
    4. Insert the needle into the vein and draw blood slowly until the vein collapses.
    5. Approximately 500 µl blood will be obtained.
    6. ACK lysing buffer was used to lyse erythrocytes.
    7. 5 ml ACK lysing buffer was added to 500 µl of murine whole blood.
    8. Incubate at room temperature for 5 min with occasional gentle shaking.
    9. Centrifuge at 400 x g for 10 min.
    10. Discard the supernatant containing lysed erythrocytes carefully.
    11. (If necessary, repeat steps B6-10.)
    12. Wash the pellet with 2 ml PBS.
    13. Centrifuge at 400 x g for 10 min, and resuspend the pellet in the 1 ml of the above mentioned medium (step A12).
    14. After ACK treatment, the blood cells that remained included white blood cells (leukocytes) and platelets.
    15.  Final leukocyte concentration was determined by hemacytometer. Approximately 1.0~2.0 x 106/ml of neutrophils will be obtained.

  3. Neutrophil activation by LPS
    1. Human neutrophils or murine leukocytes obtained from wild-type C57/BL6 mice were suspended in the above-mentioned medium (step A12).
    2. They were seeded to the 96-well plate (first plate) at a density of 1 x 104 cells per well (100 µl).
    3.  They were stimulated with LPS at indicated concentrations (2, 20, 100, and 200 µg/ml). The plates were placed in a humidified incubator at 37 °C with CO2 (5%) for 6 h.

  4. Visualization of ex vivo NETs by fluorescence microscopy
    1. Cell fixation is not performed for ex vivo live cell imaging.
    2. A cell-impermeable DNA binding dye, SYTOX Green (excitation 504/emission 523) is a 5 mM solution.
    3. To make a 10 µM SYTOX Green solution, mix 2 µl of a 5mM SYTOX Green solution with 1,000 µl PBS.
    4. 100 µl of a 10 µM SYTOX Green solution was added to each well (containing 100 µl).
    5. SYTOX Green (final concentration is 5 µM) can only get into the cell when the cell membrane was compromised. Therefore, SYTOX Green should stain both extracellular DNAs and nuclei of non-viable neutrophils.
    6. After adding SYTOX Green, both round and fibrous structures can be observed by fluorescence microscopy. SYTOX Green stained round structures are considered as nuclei of non-viable neutrophils. On the other hand, SYTOX Green stained fibrous structures are considered as extracellular DNAs which are almost identical to NETs in this protocol.

Representative data



Figure 1. Images (A, C) under fluorescence microscopy and images (B, D) under light microscopy. SYTOX Green stained NETs were released from ex vivo murine leukocytes (neutrophils) activated by LPS (A). The image of its counterpart under light microscopy (B). SYTOX Green stained NETs were released from ex vivo human neutrophils activated by LPS (C). The image of its counterpart under light microscopy (D).

Notes

  1. LPS (Escherichia coli, serotype 0111:B4) is used ad NETs inducer. Among the various serotypes (such as B5, B12, and so on), B4 would be the best with regard to the incubation time and the results obtained.
  2. To distinguish NETs from non-viable neutrophils, a staining with anti- histone, anti-neutrophil elastase or anti-myeloperoxidase antibody would be useful.

Acknowledgments

This protocol was adapted from the previously reported in Tanaka et al. (2014). This work was partly supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (KAKENHI 25462052 to K.T.).

References

  1. Tanaka, K., Koike, Y., Shimura, T., Okigami, M., Ide, S., Toiyama, Y., Okugawa, Y., Inoue, Y., Araki, T., Uchida, K., Mohri, Y., Mizoguchi, A. and Kusunoki, M. (2014). In vivo characterization of neutrophil extracellular traps in various organs of a murine sepsis model. PLoS One 9(11): e111888.

简介

中性粒细胞外陷阱(NET)是用核和颗粒蛋白(例如组蛋白,嗜中性粒细胞弹性蛋白酶或髓过氧化物酶)修饰的细胞外DNA。 它们表现出纤维状网状,网状或线状结构。 在这里,我们描述了关于从使用荧光显微镜由脂多糖(LPS)活化的嗜中性粒细胞释放的体外 NET的可视化的方案。

材料和试剂

  1. 来自野生型C57/BL6小鼠(日本SLC,Inc。)的全血
  2. 来自人类志愿者的全血
  3. LPS(大肠杆菌,血清型0111:B4)(Sigma-Aldrich,目录号:L4391)
  4. Polymorphprep TM (Axis Shield PoC AS,目录号:1114683)
  5. RPMI 1640培养基(无酚红)(Life Technologies,目录号:32404-014)
  6. 胎牛血清(Life Technologies,目录号:12483-020)
  7. ACK(氯化铵 - 钾)裂解缓冲液(Lonza,目录号:10-548E)
  8. SYTOX Green(Life Technologies,Invitrogen TM ,目录号:S7020)

设备

  1. 玻璃巴斯德吸管(Iwaki品牌,Asahi Techno Glass Corporation)
  2. 96孔板(TPP Techno Plastic Products AG)
  3. CO 2 孵化器(SANYO)
  4. 荧光显微镜(Olympus,型号:IX71)

程序

  1. 人类中性粒细胞的分离
    1. 从健康人志愿者获得静脉血(每只6ml)。
    2. 用中性密度梯度离心分离中性粒细胞 根据制造商的说明书制备多晶型物。
    3.   EDTA抗凝血(最佳浓度为1.5毫克/毫升 的血液)铺在6ml Polymorphprep溶液上并离心 在500×g下30分钟。
    4. 使用玻璃巴斯德吸管小心收获粒细胞级分。
    5. 用2ml磷酸盐缓冲液(PBS)洗涤含有粒细胞的沉淀。
    6. 在400×g离心10分钟,并将沉淀重悬在1ml PBS中。
    7. 当存在时,用ACK裂解缓冲液裂解红细胞。
    8. 将1ml ACK裂解缓冲液加入具有残余红细胞的沉淀。
    9. 在室温下孵育5分钟,偶尔吸液。
    10. 用2ml PBS洗涤含有粒细胞的沉淀。
    11. 在400×g离心10分钟。
    12. 将嗜中性粒细胞重悬浮于1ml不含酚红的补充有1%胎牛血清的RPMI 1640中。
    13. 通过血细胞计数器测定最终嗜中性粒细胞浓度。 将获得约0.5〜1.0×10 7个/ml的嗜中性粒细胞。
    14. 通过血液涂片上的May-GrünwaldGiemsa染色评价,中性粒细胞纯度被确认为常规> 90%。
    15. 简而言之,将空气干燥的涂片载玻片浸入1ml May-Grunwald溶液中1分钟。
    16. 加入等份的磷酸盐缓冲液并孵育3分钟。
    17. 倒出污渍,用自来水冲洗载玻片。
    18. 浸泡在8%Giemsa溶液中20分钟。
    19. 用自来水冲洗载玻片并风干。

  2. 鼠白细胞的分离
    1. 从麻醉的野生型C57/BL6小鼠的下腔静脉中取出肝素化的血液。
    2. 简而言之,打开腹部,识别肾脏之间的下腔静脉。
    3. 使用25号针头和1毫升注射器充满50微升肝素防止血液凝固。
    4. 将针插入静脉,并缓慢抽取血液,直到静脉塌陷。
    5. 将获得约500μl血液。
    6. ACK裂解缓冲液裂解红细胞。
    7. 将5ml ACK裂解缓冲液加入500μl鼠全血中。
    8. 在室温孵育5分钟,偶尔轻轻摇动。
    9. 在400×g离心10分钟。
    10. 小心地弃去含有裂解的红细胞的上清液。
    11. (如有必要,重复步骤B6-10。)
    12. 用2ml PBS洗涤沉淀。
    13. 在400×g离心10分钟,并将沉淀重悬于1ml上述培养基中(步骤A12)。
    14. 在ACK处理后,剩余的血细胞包括白细胞(白细胞)和血小板。
    15.  最终白细胞浓度通过血球计数器测定。 将获得约1.0〜2.0×10 6个/ml的嗜中性粒细胞。

  3. LPS引起的嗜中性粒细胞活化
    1. 从野生型获得的人嗜中性粒细胞或鼠白细胞 将C57/BL6小鼠悬浮于上述培养基中(步骤A12)。
    2. 将它们以1×10 4个细胞/孔(100μl)的密度接种到96孔板(第一个板)中。
    3.  用指定浓度的LPS刺激它们(2,20,100, 和200μg/ml)。 将平板置于37℃的潮湿培养箱中 ℃,CO 2(5%)6小时

  4. 通过荧光显微镜可视化的体外NETs。
    1. 不对离体活细胞成像进行细胞固定。
    2. 细胞不可渗透的DNA结合染料SYTOX Green(激发504 /发射523)是5mM溶液。
    3. 要制备10μMSYTOX Green溶液,将2μl5 mM SYTOX Green溶液与1,000μlPBS混合。
    4. 向每个孔(含有100μl)中加入100μl的10μMSYTOX Green溶液。
    5. SYTOX Green(终浓度为5μM)只能进入细胞 当细胞膜受损。 因此,SYTOX Green应该 染色细胞外DNA和非存活嗜中性粒细胞的核。
    6. 添加SYTOX Green后,圆形和纤维结构都可以 通过荧光显微镜观察。 SYTOX绿色染色圆 结构被认为是非存活中性粒细胞的核。 上的 另一方面,SYTOX绿色染色纤维结构被认为是 胞外DNA,其在该方案中几乎与NET相同。

代表数据



图1.荧光显微镜下的图像(A,C)和光学显微镜下的图像(B,D)。 SYTOX绿色染色的NETs从离体小鼠白细胞 )被LPS激活(A)。 在光学显微镜下其对应物的图像(B)。 SYTOX绿色染色的NETs从离体的由LPS激活的人嗜中性粒细胞释放(C)。 其在光学显微镜(D)下的对应物的图像。

笔记

  1. LPS(大肠杆菌,血清型0111:B4)用于NETs诱导物。 在各种血清型(例如B5,B12等)中,B4对于温育时间和获得的结果将是最好的。
  2. 为了区分NETs和非存活中性粒细胞,用抗组蛋白,抗中性粒细胞弹性蛋白酶或抗髓过氧化物酶抗体染色将是有用的。

致谢

该协议改编自以前在Tanaka等人(2014)中报道的。 这项工作部分得到了日本教育,文化,体育,科学和技术部赠款(KAKENHI 25462052至K.T.)的支持。

参考文献

  1. Tanaka,K.,Koike,Y.,Shimura,T.,Okigami,M.,Ide,S.,Toiyama,Y.,Okugawa,Y.,Inoue,Y.,Araki,T.,Uchida, Mohri,Y.,Mizoguchi,A.and Kusunoki,M.(2014)。 表征小鼠脓毒症的各种器官中的中性粒细胞胞外陷阱 模型。 PLoS One 9(11):e111888。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tanaka, K., Shimura, T., Toiyama, Y., Okugawa, Y., Inoue, Y., Araki, T., Mohri, Y., Mizoguchi, A. and Kusunoki, M. (2015). Visualization of ex vivo Neutrophil Extracellular Traps by Fluorescence Microscopy. Bio-protocol 5(15): e1550. DOI: 10.21769/BioProtoc.1550.
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