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Quantification of ex vivo Neutrophil Extracellular Traps
通过DNA对体外中性粒细胞的胞外杀菌网进行量化   

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Abstract

Neutrophil extracellular traps (NETs) are fibrous mesh-like, web-like, or string-like structures which are composed of DNA, histones, and granule proteins such as neutrophil elastase or myeloperoxidase. When activated by phorbol myristate acetate, interleukin-8, lipopolysaccharide (LPS), and various pathogens, neutrophils release NETs. We reported that NETs were classified as two distinct forms; cell-free NETs that were released away from neutrophils and anchored NETs that were anchored to neutrophils. In general, extracellular DNAs are used as a surrogate marker of NETs. Here, we describe a protocol regarding quantitative procedures of extracellular DNAs released from ex vivo neutrophils activated by LPS using fluorometric double-stranded DNA (dsDNA) quantification assay.

Materials and Reagents

  1. Whole blood from wild-type C57/BL6 mice (Japan SLC)
  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. Quant-iTTM PicoGreen dsDNA Assay Kit (Life Technologies, InvitrogenTM, catalog number: P11496 )

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 microplate reader (Perkin Elmer, model: 2030ARVOX )

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 1ml 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. Quantification of cell-free NETs
    1. Since cell-free NETs were released away from neutrophils, they were quantified as extracellular DNAs in culture supernatants.
    2. The culture supernatant of each well (100 µl) was collected and directly transferred to another 96-well plate (second plate). A centrifugation is not needed.
    3. Cell-free NETs in culture supernatants were quantified as dsDNA in the culture supernatants using a Quant-iTTM PicoGreen dsDNA Assay Kit.
    4. In brief, an equal volume of PicoGreen reagent (100 µl) was added to each well containing culture supernatants (100 µl).
    5. Plates were incubated for 5 min at room temperature with protecting from light.
    6. The PicoGreen dye that was bound to dsDNA was measured using a fluorescence microplate reader. The following settings were used to read the PicoGreen results: Excitation wavelength and bandwidth were 485 nm and 14 nm, respectively. Emission wavelength and bandwidth were 535 nm and 25 nm, respectively.
    7. The DNA concentration was calculated using a standard curve generated from a series of Lambda DNA standard (100 µg/ml) provided by the manufacturer.

  5. Quantification of anchored NETs
    1. To quantify anchored NETs that were anchored to neutrophils, the first plate was used after removal of culture supernatants.
    2.  Fresh medium (100 µl) was added to each well of the first plate. An equal volume of PicoGreen reagent (100 µl) was added to each well to quantify anchored NETs.
    3. Plates were incubated for 5 min at room temperature protected from light.
    4. The PicoGreen dye that was bound to dsDNA was measured using a fluorescence microplate reader.
    5.  The DNA concentration was calculated using a standard curve generated from a series of Lambda DNA standard (100 µg/ml) provided by the manufacturer.

Representative data



Figure 1. The representative data of ex vivo NETs released from human neutrophils activated by LPS. (A). The DNA concentration at the indicated dose of LPS was shown as the sum of the amount of anchored and cell-free NETs. (B). The DNA concentration of anchored NETs was shown at the indicated dose of LPS on the first plate. Anchored NETs were anchored to neutrophils adhering to the plate. (C). The DNA concentration of cell-free NETs was shown at the indicated dose of LPS. They were transferred to the second plate as the supernatant of the first plate.

Notes

Gentle aspiration of the supernatant should be needed to avoid the contamination of anchored NETs into the second plate.

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)是由DNA,组蛋白和颗粒蛋白如嗜中性粒细胞弹性蛋白酶或髓过氧化物酶组成的纤维状网状,网状或绳状结构。 当被佛波醇肉豆蔻酸乙酯,白细胞介素-8,脂多糖(LPS)和各种病原体激活时,嗜中性粒细胞释放NETs。 我们报告,NETs被分为两种不同的形式; 从嗜中性粒细胞释放的无细胞NETs和锚定于嗜中性粒细胞的锚定NET。 通常,细胞外DNA用作NETs的替代标记。 在这里,我们描述一个议定书关于使用荧光双链DNA(dsDNA)定量测定由LPS激活的离体嗜中性粒细胞释放的细胞外DNA的定量程序。

材料和试剂

  1. 来自野生型C57/BL6小鼠(日本SLC)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. Quant-iT TM PicoGreen dsDNA测定试剂盒(Life Technologies,Invitrogen TM ,目录号:P11496)

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. 荧光酶标仪(Perkin Elmer,型号:2030ARVOX)

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. 通过血细胞计数器测定最终白细胞浓度。 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. 它们用指定浓度的LPS刺激(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. 无细胞NET的定量
    1. 因为无细胞的NETs被释放出中性粒细胞,他们是 定量为培养上清液中的细胞外DNA。
    2. 的 收集每孔的培养上清液(100μl)并直接 转移到另一个96孔板(第二板)。 离心是   不需要。
    3. 培养上清液中的无细胞NETs 使用Quant-iT TM PicoGreen dsDNA测定试剂盒在培养物上清液中定量为dsDNA。
    4. 简言之,将等体积的PicoGreen试剂(100μl)加入到含有培养上清液(100μl)的每个孔中。
    5. 将板在室温下避光孵育5分钟。
    6. 结合dsDNA的PicoGreen染料使用a 荧光酶标仪。 使用以下设置读取   PicoGreen结果:激发波长和带宽为485 nm 和14nm。 发射波长和带宽为535nm 和25nm。
    7. 计算DNA浓度 使用从一系列λDNA标准产生的标准曲线 (100μg/ml)。

  5. 锚定NET的量化
    1. 为了定量锚定到嗜中性粒细胞的锚定的NET,在除去培养上清液后使用第一个平板。
    2.  向第一个板的每个孔中加入新鲜培养基(100μl)。 一个 将等体积的PicoGreen试剂(100μl)加入到每个孔中 量化锚定的NETs。
    3. 将平板在室温避光保温5分钟。
    4. 使用荧光酶标仪测量与dsDNA结合的PicoGreen染料。
    5. >使用产生的标准曲线计算DNA浓度 从一系列λDNA标准品(100μg/ml)提供 制造商。

代表数据



图1.从由LPS激活的人嗜中性粒细胞释放的离体NETs的代表性数据。(A)。 在指定剂量的LPS的DNA浓度显示为锚定和无细胞NETs的量的总和。 (B)。 锚定的NETs的DNA浓度显示在第一个平板上的指定剂量的LPS。 锚定的NETs锚定在粘附在板上的嗜中性粒细胞上。 (C)。 在指定剂量的LPS下显示无细胞NETs的DNA浓度。 将它们作为第一板的上清液转移到第二板。

笔记

应该需要温和地吸出上清液以避免锚定的NETs污染到第二板中。

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., Okigami, M., Toiyama, Y., Okugawa, Y., Inoue, Y., Araki, T., Mohri, Y., Mizoguchi, A. and Kusunoki, M. (2015). Quantification of ex vivo Neutrophil Extracellular Traps. Bio-protocol 5(15): e1549. DOI: 10.21769/BioProtoc.1549.
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