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Aug 2020
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Neutral Comet Assay to Detect and Quantitate DNA Double-Strand Breaks in Hematopoietic Stem Cells
检测和定量造血干细胞DNA双链断裂的中性彗星试验   

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Abstract

In vertebrates, hematopoietic stem cells (HSCs) regulate the supply of blood cells throughout the lifetime and help to maintain homeostasis. Due to their long lifespan, genetic integrity is paramount for these cells, and accordingly, a number of stem cell-specific mechanisms are employed. However, HSCs tend to show more DNA damage with increasing age due to an imbalance between proliferation rates and DNA damage responses. The comet assay is the most common and reliable method to study DNA strand breaks at the single-cell level. This procedure is based on the electrophoresis of agarose-embedded lysed cells. Following the electrophoretic mobilization of DNA, it is stained with fluorescent DNA-binding dye. Broken DNA strands migrate based on fragment size and form a tail-like structure called “the comet,” whereas intact nuclear DNA remains a part of the head of the comet. Since the alkaline comet assay fails to differentiate between single and double-strand breaks (DSBs), we used a neutral comet assay to quantitate the DSBs in HSCs upon aging and other physiological stresses. The protocol presented here provides procedural details on this highly sensitive, rapid, and cost-effective assay, which can be used for rare populations of cells such as HSCs.


Graphical abstract:



The neutral comet assay is an extremely useful tool that allows the detection and quantitation of double-strand DNA breaks at the single-cell level. The graphical abstract represents a flowchart for the neutral comet assay procedure.


Keywords: Hematopoietic stem cells (造血干细胞), DNA damage (DNA损伤), Double-strand breaks (双链断裂), Neutral comet assay (中性彗星试验)

Background

During every round of cell proliferation, the genomic content is faithfully replicated, albeit with some errors. These errors lead to changes or modifications in the structure and chemical nature of genomic DNA. The replication machinery is tightly associated with DNA damage repair (DDR) pathways, thereby recovering cells from such damage (Chatterjee and Walker, 2017). Alternatively, the DNA damage response pathways can induce apoptosis (Nowsheen and Yang, 2012); however, some DNA damage is undetected and unrepaired, accumulating over a period of time. Accumulation of this DNA damage during aging poses a serious challenge to the healthy functioning of a variety of tissues, including hematopoietic systems. Recent studies have indicated that accumulation of age-related physiological changes in the stem cell population leads to alterations in immune system function (Oh et al., 2014). In addition, several age-associated malignancies have been linked to DNA damage accumulation in the stem cell compartment (Beerman, 2017). Hematopoietic stem cells have cytoprotective and genoprotective mechanisms to safeguard their long-lasting functional potential; however, it has been demonstrated that DNA damage accumulates in HSCs during the aging process. Several research groups have proposed that this accumulation is due to increased proliferation rates in old HSCs. In contrast, others show that long-term dormancy may prevent rare DNA damage from accumulating due to lack of replication-associated repair (Beerman et al., 2014).


To understand the fundamental processes involved in DNA damage accumulation and repair, quantitation of DNA damage is vital (Lu et al., 2017). In 1984, Ostling and Johanson demonstrated the electrophoretic mobility of DNA fragments from the core nuclei (Ostling and Johanson, 1984). When single cells embedded in low-melting-point agarose are electrophoresed, the DNA-damaged fragments move faster, giving rise to tail-like structures resembling a comet. The intact part of the DNA is called the head of the comet, and the trail produced due to impaired DNA is known as the tail of the comet. This single-cell gel electrophoresis assay, also known as the comet assay, is one of the most reliable and commonly used methods for analyzing DNA damage in cells. It has been successfully applied to understand the processes involved in genotoxicity, molecular epidemiology, and human bio-monitoring, along with solving basic biology questions regarding DNA damage and repair (Collins, 2004). This technique was further modified by Singh et al., who showed a substantial increase in reproducibility under alkaline conditions (Lu et al., 2017). At alkaline pH, the sensitive sites are knocked down and the strands break due to distortion in their conformation. Therefore, the alkaline comet assay is commonly used to analyze strand breaks, DNA-protein crosslinks, and alkali-labile sites; nevertheless, the specificity for double-strand breaks is compromised (Lu et al., 2017). To overcome this issue, the neutral comet assay was further developed (Calini et al., 2002); it protects the DNA double strands from distortion, thus aiding in the specific detection of double-strand DNA damage. These DNA strand breaks result in the lengthening of DNA loops in genomic DNA, which form a comet-like tail in an electrophoretic mobility assay (Cortes-Gutierrez et al., 2012). Because double-strand DNA breaks lead to mutations and chromosomal abnormalities, it is important to detect and quantitate them. The neutral comet assay is of immense importance and is highly efficient for in vivo and ex vivo systems (Sharma et al., 2011). The comets formed due to electrophoretic mobility can be visualized by fluorescence microscopy. The percentage of DNA damage can be estimated from the percentage of DNA present in the comet tail since they are directly proportional to one other. With advancements in technology such as fluorescent staining and automated comet scoring software, the comet assay has become popular for detecting DNA damage (Beedanagari, 2017). In the case of rare cell populations such as HSCs, the neutral comet assay has emerged as a promising technique to study DNA damage responses due to physiological, pathological, and age-associated stresses (Hazlehurst and Dalton, 2001; Milyavsky et al., 2010). The present protocol describes the method to assess DNA damage in freshly sorted hematopoietic stem cells from mice bone marrow.

Materials and Reagents

  1. Autoclaved lint-free tissue paper

  2. 60-mm tissue culture plates (Eppendorf, catalog number: 0030701119)

  3. 26 G needle (BD PrecisionGlide, BD Medical (S) Pvt Ltd, catalog number: 302806)

  4. 1-ml syringe (BD Medical (S) Pvt Ltd, catalog number: 303060)

  5. 40-µm nylon strainer (Falcon, catalog number: 352340)

  6. Bone marrow cells from mice

  7. FACS tubes (Falcon, catalog number: 352054)

  8. Comet slides (CometSlidesTM 20 well, Trevigen, catalog number: 4252-02K-01)

  9. MACS apparatus

    1. MS column (MACS, Miltenyi Biotec, catalog number: 130-042-201)

    2. MACS multistand (MACS, Miltenyi Biotec, catalog number: 002139)

    3. MiniMACSTM separator (MACS, Miltenyi Biotec, catalog number: 130-042-102)

  10. Antibodies

    1. APC-conjugated lineage antibody cocktail [2:100 concentration] (BD PharmingenTM, BD Biosciences, catalog number: 51-9003632)

    2. Anti-mouse c-kit PE [1:100 concentration] (Biolegend, catalog number: 105807)

    3. Anti-mouse Sca-1 PerCP-Cy5.5 [1:100 concentration] (Biolegend, catalog number: 122524)

  11. 1% agarose (Sigma-Aldrich, catalog number: A9539)

  12. 0.75% low-melting-point agarose (LM agarose) (Sigma-Aldrich, catalog number: A9414)

  13. 70% ethanol (Changshu Hongsheng Fine Chemical Co., Ltd, catalog number: 1170)

  14. SYBR® Gold staining solution (ThermoFisher Scientific, Invitrogen, catalog number: S33102)

  15. 10× RBC lysis buffer (Biolegend, catalog number: 420301)

  16. Lysis buffer (see Recipes)

  17. 1× neutral electrophoresis buffer (see Recipes)

  18. DNA precipitation solution (see Recipes)

  19. 1× phosphate-buffered saline (PBS) (see Recipes)

Equipment

  1. Electrophoresis unit (Mini-Sub® Cell GT, Bio-Rad, catalog number: 329BR014542)

  2. Epifluorescence microscope (Olympus IX2-ILL100, OLYMPUS CORPORATION, catalog number: 0M04213)

  3. Brightfield microscope (Primovert, Ziess, catalog number: 3842010199)

Software

  1. ImageJ – Open Comet (Benjamin M. Gyori and Gireedhar Venkatachalam, www.cometbio.org)

  2. Excel 2016 (Microsoft Corporation)

  3. GraphPad Prism (GraphPad Software, Inc., www.graphpad.com)

Procedure

  1. Cell preparation

    1. Euthanize a C57Bl6/J mouse, dissect out the hind limbs, and remove the extra tissue from the bones to separate the femur and tibia.

    2. Flush the bone marrow cells into a 50-ml polypropylene tube using autoclaved 1× PBS and a 26 G needle with a 1-ml syringe until the bones are cleared inside.

    3. Mix the cells to make a single cell suspension and strain through a 40-μm nylon strainer. Centrifuge at 600 × g for 5 min at 4°C.

    4. Lyse the RBCs by adding 500 µl 1× RBC lysis buffer (diluted in 1× PBS) to the pellet and neutralize the reaction after 2 min by adding 2 ml 1× PBS. Centrifuge at 600 × g for 5 min at 4°C.

    5. Resuspend the cell pellet in 1 ml 1× PBS and count the cells using a hemocytometer.

    6. Incubate the cells with the required antibodies (2 µl/5 million cells for Lin APC and 1 µl/5 million cells for c-Kit PE and Sca-1 PerCP-Cy5.5) to stain hematopoietic progenitor cells (Lin APC cocktail, c-Kit PE, Sca-1 PerCP-Cy5.5) for 30 min.

    7. Centrifuge at 600 × g for 5 min at 4°C. Wash the cells with 1× PBS and transfer to a 5-ml FACS tube for sorting.

    8. Sort the pure LSK (Lineage Sca-1+ ckit+) population into 1× PBS and store at 4°C until further use.

      Note: Alternatively, MACS sorting can be used to isolate less pure HSC progenitor populations.


  2. Slide preparation

    1. Weigh 1 g agarose and dissolve in 100 ml 1× PBS using a microwave oven.

    2. Place the comet slides inside a plastic container in a slanted position (Figure 1A). Pipette out 1 ml agarose and evenly coat the slides and, leave to solidify for 10-15 min at room temperature (Figure 1B).

    3. Freshly prepare 0.75% low-melting (LM) agarose using 1× PBS in a 100-ml beaker. Dissolve using a microwave oven.

      Note: Transfer the molten 0.75% LM agarose to a storage bottle for further use. Before use, re-melt in a beaker containing hot water.

    4. Allow the hot LM agarose to cool for at least 20 min at 37°C.

    5. Add 1 × 105 cells/ml to the molten LM agarose at a ratio of 1:10 (v/v).

      Note: If you take 500 µl molten LM agarose, add 50 µl cells in 1× PBS (Ca2+ and Mg2+ free) at 1 × 105 cells/ml.

    6. Immediately pipette 50 µl suspension onto the comet slide. Use the tip of the pipette to spread the mixture uniformly over the surface area and allow to solidify (Figure 1C).

      Note: If the sample mixture is not dispersing uniformly on the slide, heat up the LM agarose at 37°C before suspending the cells, and prepare the sample mixture again.

    7. Keep the slides at 4°C for 10 min in the dark (e.g., place in a refrigerator).

      Note: A clear 0.5 mm ring appears at the edge of the well (Figure 1D). To increase the adherence of the sample to the slide, the gelling time and humidity should be increased.



      Figure 1. Preparation of a comet slide. A. A clean Trevigen® CometSlideTM HT is placed inside a plastic container. B. The comet slide is pre-coated with molten 1% agarose. This ensures that cells adhere to the slide efficiently. After pre-coating with agarose, allow the gel to solidify. C. The 0.75% LM agarose containing the cells is added gradually to the slide. Allow the gel to solidify at 4°C. D. The comet slide after solidification of the gel.


  3. Lysis and Electrophoresis

    1. Prepare the lysis solution (see Recipes) and place on ice for at least 20 min before use.

    2. Pour the required amount of pre-chilled lysis solution in a tray and immerse the slides. Place the tray on ice or at 4°C for 1 h.

    3. Take the slides from the lysis solution and drain the excess.

    4. Wash the slides by placing them in 50 ml pre-chilled 1× neutral electrophoresis buffer (see Recipes) at 4°C for 30 min.

    5. Pour 950 ml pre-chilled 1× neutral electrophoresis buffer into the Comet Assay® ES tank. Place the slides in the electrophoresis slide tray and cover with the slide tray overlay (Figure 2A).

    6. Set the power supply to 21 volts and perform electrophoresis for 1 h at 4°C.

    7. For other electrophoresis units, align the slides equidistant from the electrodes, add 1× neutral electrophoresis buffer not exceeding 0.5 cm above the slide (Figure 2B), and apply voltage for 30 min at 1 volt per cm, measuring the distance between the two opposite electrodes of the electrophoresis tank (e.g., If the distance is 21 cm then apply 21 V).

    8. Run the electrophoresis unit either in a cold room or over an ice tray containing enough ice to partially cover the unit (Figure 2C).



      Figure 2. Electrophoresis under neutral conditions. A. After lysis, the comet slide is placed in the electrophoretic unit and neutral electrophoresis buffer is added to the buffer tank. B. The neutral electrophoresis buffer should not exceed 0.5 cm above the slide. C. Perform electrophoresis at 21 volts for 1 h at 4°C.


  4. Staining and visualization of slides

    1. Drain the excess neutral electrophoresis buffer and immerse the slides in DNA precipitation solution for 30 min at room temperature (see Recipes).

    2. Remove the slides and immerse them in 70% ethanol for 5 min at room temperature.

    3. Allow the samples to dry for 10-15 min at room temperature.

      Note: Drying the samples will allow the cells to settle in a single plane, making visualization, recording, and quantitation easier. Samples can be stored with desiccant at room temperature before scoring.

    4. Add 100 µl diluted SYBR® Gold onto each sample and incubate for 30 min.

      Note: Trevigen provides the CometAssay® Silver Staining Kit designed for comet staining. Silver staining helps in the visualization of comets on any transmission light microscope and permanently stains the samples for long-term storage. It is recommended that samples are dried before silver staining.

    5. Tap the slides gently to remove excess SYBR® Gold solution. Allow the slide to dry completely at room temperature in the dark.

    6. View the slides using epifluorescence microscopy and obtain unbiased images for every cell that could be visualized.

      Note: The efficiency of the method can be tested using cells challenged with genotoxic stress as a positive control. Here, hematopoietic stem and progenitor cells from young adults (8 weeks old) (Figure 3A) were compared with cells from aged mice (2 years old) (Figure 3B). Comet tail formation in cells from aged mice is clearly visible. Comparison between the two cell types for the proportion of genomic DNA in the head versus the tail can be used as a reliable quantitation of the extent of DNA damage.



      Figure 3. Staining and visualization of cells. A. HSC from young mice; the nucleus in the cell is intact, indicating that there is no DNA damage. B. HSC from old mice; the nucleus in the cell is not intact and is forming a head and a comet tail, indicating that there is DNA damage.


  5. Scoring and analysis of the cells

    1. Scoring of the comet is performed in the ImageJ plugin – Open Comet. Launch the plugin in ImageJ (Figure 4A).

    2. Input your image files to the Input files option by clicking on the Browse button (Figure 4B).

    3. Choose an Output directory where the output files will be stored, along with the name of the file that it should be saved under (Figure 4C and 4D).



      Figure 4. Comet scoring using ImageJ. A. Open the ImageJ software and click on Plugins. Select OpenComet_src_v1.3.1 and click on OpenComet. B. A dialogue box will open, where you need to browse the Input files and Output directory. C. Select the images for scoring the input file. D. Choose the Output directory.


    4. Choose the default analysis option and Click RUN.

    5. The analyzed output data will appear in the Output directory.

    6. The Output file consists of an Output Image and an Output Spreadsheet. Review all the Output images for false analysis and outliers. Note the Number given specifically for each image (Figure 5A).

    7. Open the Spreadsheet and manually delete the outlier numbers.

    8. Obtain the values for Olive moment and Tail moment from the Spreadsheet and graphically represent the data using GraphPad PRISM (Figure 5B).



      Figure 5. Results of comet scoring. A. Identification of the comet head and tail. B. Excel spreadsheet containing the computed comet parameters.

Recipes

  1. Lysis buffer (Ingredients/1,000 ml)

    3.5 M NaCl – 146.1 g

    100 mM EDTA – 37.2 g

    100 mM Trizma Base – 1.2 g

    Dissolve the ingredients in 700 ml dH2O.

    Add 8 g NaOH pellets and adjust the pH to 10.0 using HCl or NaOH (concentrated).

    50 ml is required for each assay.

    Add 1% Triton X-100 – 0.5 ml.

    10% dimethyl sulphoxide (DMSO) – 5 ml

  2. 1× neutral electrophoresis buffer

    To prepare 10× neutral electrophoresis buffer:

    Tris base (mol. wt. = 121.14) – 60.57 g

    Sodium acetate (mol. wt. = 136.08) – 204.12 g

    Dissolve in 450 ml dH2O and adjust the pH to 9.0 using glacial acetic acid.

    Adjust the volume to 500 ml and filter sterilize. Store at room temperature.

    Dilute the 10× stock to 1× in dH2O to prepare 1 L working strength buffer and pre-chill at 4°C.

  3. DNA precipitation solution

    Prepare a 10 ml stock solution of 7.5 M ammonium acetate: NH4Ac (mol. wt. = 77.08) = 5.78 g

    dH2O (after NH4Ac is dissolved) – 10 ml

    For 50 ml DNA precipitation solution:

    7.5 M NH4Ac (mol. wt. = 77.08) – 6.7 ml

    95% EtOH (reagent grade) – 43.3 ml

  4. SYBR® Gold staining solution

    From SYBR® Gold concentrate (10,000× in DMSO)

    SYBR® Gold – 1 µl

    Electrophoresis Buffer – 10 ml

    Store at 4°C in the dark

  5. 1× phosphate-buffered saline (PBS)

    To prepare 10× PBS stock solution:

    NaCl – 80 g

    KCl – 2 g

    Na2HPO4 – 14.4 g

    KH2PO4 – 2.4 g

    Take 800 ml Milli-Q water, dissolve the salts and adjust the pH to 7.4, and make up the volume to 1,000 ml

    Dilute the 10× stock to 1× in dH2O

Acknowledgments

This work was supported by the Wellcome Trust/DBT India Alliance Fellowship (IA/I/15/2/502061) awarded to SK and intramural funds from the Indian Institute of Science Education and Research Thiruvananthapuram (IISER TVM). The institutional animal facility is supported by funds from the Department of Science and Technology, Government of India (under FIST scheme; SR/FST/LS-II/2018/217). IMR is supported by a Senior Research Fellowship from the University Grants Commission (UGC), India. This protocol was taken from the article by Biswas et al. (2020) “The Periostin/Integrin-av Axis Regulates the Size of Hematopoietic Stem Cell Pool in the Fetal Liver” and was modified from the protocol described in the Trevigen comet assay kit (Biswas et al., 2020).

Competing interests

The authors have no competing financial or non-financial interests relevant to this study.

Ethics

The animals used to isolate the HSCs for DNA damage analysis (C57BL/6J-CD45.2) were bred and maintained in the animal facility at IISER Thiruvananthapuram. The animals were maintained as per the guidelines provided by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forests (MoEF), Government of India. During the experiments, mice were maintained in isolator cages and allowed access to autoclaved acidified water and irradiated food ad libitum. All animal experiments were approved by the Institutional Animal Ethics Committee (IAEC) of the institute.

References

  1. Beedanagari, S. (2017). 4.11-Genetic Toxicology. In: Chackalamannil, S., Rotella, D. and Ward, S. E. (Eds.). Comprehensive Medicinal Chemistry III. pp 195-203. Elsevier, Oxford.
  2. Beerman, I. (2017). Accumulation of DNA damage in the aged hematopoietic stem cell compartment. Semin Hematol 54(1): 12-18.
  3. Beerman, I., Seita, J., Inlay, M. A., Weissman, I. L. and Rossi, D. J. (2014). Quiescent hematopoietic stem cells accumulate DNA damage during aging that is repaired upon entry into cell cycle. Cell Stem Cell 15(1): 37-50.
  4. Biswas, A., Roy, I. M., Babu, P. C., Manesia, J., Schouteden, S., Vijayakurup, V., Anto, R. J., Huelsken, J., Lacy-Hulbert, A., Verfaillie, C. M. and Khurana, S. (2020). The Periostin/Integrin-alphav Axis Regulates the Size of Hematopoietic Stem Cell Pool in the Fetal Liver. Stem Cell Reports 15(2): 340-357.
  5. Calini, V., Urani, C. and Camatini, M. (2002). Comet assay evaluation of DNA single- and double-strand breaks induction and repair in C3H10T1/2 cells. Cell Biol Toxicol 18(6): 369-379.
  6. Chatterjee, N. and Walker, G. C. (2017). Mechanisms of DNA damage, repair, and mutagenesis. Environ Mol Mutagen 58(5): 235-263.
  7. Collins, A. R. (2004). The comet assay for DNA damage and repair: principles, applications, and limitations. Mol Biotechnol 26(3): 249-261.
  8. Cortes-Gutierrez, E. I., Hernandez-Garza, F., Garcia-Perez, J. O., Davila-Rodriguez, M. I., Aguado-Barrera, M. E. and Cerda-Flores, R. M. (2012). Evaluation of DNA single and double strand breaks in women with cervical neoplasia based on alkaline and neutral comet assay techniques. J Biomed Biotechnol 2012: 385245.
  9. Hazlehurst, L. A. and Dalton, W. S. (2001). Mechanisms associated with cell adhesion mediated drug resistance (CAM-DR) in hematopoietic malignancies. Cancer Metastasis Rev 20(1-2): 43-50.
  10. Lu, Y., Liu, Y. and Yang, C. (2017). Evaluating In Vitro DNA Damage Using Comet Assay. J Vis Exp(128).
  11. Milyavsky, M., Gan, O. I., Trottier, M., Komosa, M., Tabach, O., Notta, F., Lechman, E., Hermans, K. G., Eppert, K., Konovalova, Z., Ornatsky, O., Domany, E., Meyn, M. S. and Dick, J. E. (2010). A distinctive DNA damage response in human hematopoietic stem cells reveals an apoptosis-independent role for p53 in self-renewal. Cell Stem Cell 7(2): 186-197.
  12. Nowsheen, S. and Yang, E. S. (2012). The intersection between DNA damage response and cell death pathways. Exp Oncol 34(3): 243-254.
  13. Oh, J., Lee, Y. D. and Wagers, A. J. (2014). Stem cell aging: mechanisms, regulators and therapeutic opportunities. Nat Med 20(8): 870-880.
  14. Ostling, O. and Johanson, K. J. (1984). Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123(1): 291-298.
  15. Sharma, A., Shukla, A. K., Mishra, M. and Chowdhuri, D. K. (2011). Validation and application of Drosophila melanogaster as an in vivo model for the detection of double strand breaks by neutral Comet assay. Mutat Res 721(2): 142-146.

简介

[摘要]在vertebrat ES,造血干细胞(HSC)调节血细胞的整个寿命和帮助供应到保持动态平衡。由于其寿命长,遗传完整性是极为重要的这些细胞并且因此,一些干细胞-特异性的机制被采用。然而,由于增殖率和 DNA 损伤反应之间的不平衡,HSC 随着年龄的增长往往表现出更多的 DNA 损伤。与c OMET测定法是最常见和最可靠的方法在研究DNA链断裂的单-细胞水平。这个程序是基于该 琼脂糖电泳-嵌入的裂解细胞。下列的DNA的电泳动员,它沾有荧光DNA -结合的染料。断裂的 DNA 链根据片段大小迁移并形成称为“彗星”的尾状结构,而完整的核 DNA 仍然是彗星头部的一部分。由于碱性彗星试验失败单和双链断裂(DSB的)之间进行区分,我们使用一个中性彗星实验孔定量泰特的在老化和其他生理应力在HSC中的DNA双链断裂。这里介绍的协议提供了有关该高度敏感,快速的程序细节,和具有成本效益分析,其可用于稀有人口小号细胞如造血干细胞。

图形概要:

所述n eutral彗星试验是一个非常有用的工具,它允许所述检测和孔定量吨在双链DNA断裂的通货膨胀的单-细胞水平。图形抽象表示一个流程图为中性彗星测定程序。

[背景] 在每一轮细胞增殖过程中,基因组内容都被忠实地复制,尽管存在一些错误。这些错误会导致基因组 DNA 的结构和化学性质发生变化或修改。第r eplication机械被紧密地与DNA损伤修复(DDR)途径有关,从而回收细胞免受这类损伤(Chatterjee的和Walker,2017)。备选地,DNA损伤反应途径可诱导apopto SIS (Nowsheen和Yang,2012); ħ H但是,一些DNA损伤是未检测到的未修理和,积累在一段时间。甲的第ccumulation是DNA损伤期间老化姿态š到的各种组织,包括造血系统的健康运转的严重挑战。最近的研究我NDI cated的积累的年龄-在免疫系统功能有关的生理变化的干细胞群导致改变(哦,等,2014)。此外,一些年龄-相关的恶性肿瘤已链接到干细胞室(Beerman,2017)DNA损伤的积累。造血干细胞具有细胞保护和genoprotective机制来保障他们的长-持久的功能潜力; ħ H但是,已经证明吨帽子DNA损伤积累小号在老化过程中在HSC中。几个研究小组提出,日是积累是由于增加proliferat离子在老造血干细胞率。在C ontra ST ,其他人显示,长期休眠可防止罕见DNA损伤从accumulat荷兰国际集团由于缺乏复制- (Beerman相关修复。等人,2014)。

为了理解参与DNA损伤积累和修复的基本过程,孔定量吨DNA损伤的通货膨胀是至关重要的(路等人,2017)。1984 年,Ostling 和 Johanson 证明了来自核心核的 DNA 片段的电泳迁移率(Ostling 和 Johanson,1984)。当嵌入在低的单细胞-熔化琼脂糖电泳被,将DNA -损伤片段移动速度更快,从而产生尾-样结构类似于彗星。DNA 的完整部分被称为彗星的头部,由于DNA 受损而产生的轨迹被称为彗星的尾部。这种单-细胞凝胶电泳,也被称为彗星试验,是对ANALY最可靠和最常用的方法之一ž荷兰国际集团在细胞的DNA损伤小号。它已成功应用于了解涉及基因毒性、分子流行病学和人类生物监测的过程,以及解决有关DNA 损伤和修复的基本生物学问题(柯林斯,2004 年)。Ť他的技术是由辛格进一步修饰等。,谁发现一个在再现性在碱性条件下显着增加小号(路等人,2017)。在碱性 pH 值下,敏感位点被击倒,并且由于其构象扭曲而导致链断裂。因此,该碱性彗星试验常用于ANALY Ž È链断裂,DNA蛋白质交联,和碱-不稳定的位点; 尽管如此,双链断裂的特异性受到了损害(Lu等人,2017 年)。为了克服这个问题,进一步开发了中性彗星试验(Calini et al. , 2002),它可以保护 DNA 双链免受扭曲,从而有助于双链 DNA 损伤的特异性检测。这些DNA链断裂导致DNA的延长在基因组DNA环,其形成在电泳迁移率试验(彗星状尾巴科尔特斯-Gutierrez的等人,2012)。由于双链DNA断裂导致突变小号和染色体异常,它来检测和孔定量是很重要的泰特他们。所述n eutral彗星测定法是极为重要和是高度高效的体内和体外系统(夏尔马等人,2011)。由于形成为电泳迁移率的彗星可以可视化通过荧光显微镜ÿ 。DNA损伤的百分比可以从DNA的百分比来估计本在彗星尾巴因为它们是直接正比于一个其他。随着技术的进步,例如作为荧光染色和自动化彗星得分软件,该彗星试验已成为流行用于检测DNA损伤(Beedanagari,2017)。在罕见的情况下,细胞群,如造血干细胞,在中性彗星试验已经成为一种有希望的技术来研究DNA损伤应答由于生理,病理,和年龄相关的应力(Hazlehurst的和道尔顿,2001; Milyavsky等人,2010 )。所述PRES ENT协议描述来评估DNA损伤在新鲜分选的来自小鼠的骨髓造血干细胞的方法。

关键字:造血干细胞, DNA损伤, 双链断裂, 中性彗星试验

材料和试剂
 
蒸压皮棉-免费纸巾
60 -毫米的组织培养板小号仪(Eppendorf,目录号:0030701119)
26 G 针(BD PrecisionGlide,BD Medical(S)Pvt Ltd,目录号:302806)
1 - ml注射器(BD Medical(S)Pvt Ltd,目录号:303060)
40 - μm尼龙过滤器(F爱尔康,Ç atalog号:352340)
小鼠骨髓细胞
FACS 管(F alcon ,目录号:352054)
彗星滑梯(CometSlides TM 20 well,Trevigen,目录号:4252-02K-01)
MACS设备
MS 柱(MACS,Miltenyi Biotec,目录号:130-042-201)
MACS multistand(MACS,Miltenyi Biotec,目录号:002139)
应用MiniMACS TM小号eparator(MACS,美天旎,目录号:130-042-102)
抗体
APC -共轭升ineage抗体混合物[2:100浓度(BD Pharmingen公司TM ,BD Biosciences公司,目录号:51-9003632)
抗小鼠 c-kit PE [1:100 浓度](Biolegend,目录号:105807)
抗小鼠 Sca-1 PerCP-Cy5.5 [1:100 浓度](Biolegend,目录号:122524)
1%一garose(S IGMA -A ldrich ,目录号:A9539)
0.75%升流-熔化琼脂糖(LM琼脂糖)(Sigma-Aldrich公司,目录号:A9414)
70%ë THANOL(常熟宏盛精细化工有限公司,目录号:1170)
SYBR ®金染色溶液(赛默飞世科学,Invitrogen公司,目录编号:S33102)
10 × RBC升ysis b uffer(Biolegend公司,目录号:420301)
裂解缓冲液(见配方)
1× n中性电泳缓冲液(见配方)
DNA沉淀溶液(见配方)
1 × p磷酸盐-缓冲盐水(PBS)(见食谱)
 
设备
 
电泳单元(迷你子®细胞GT,Bio-Rad公司,目录号:329BR014542)
落射荧光显微镜(Olympus IX2-ILL100,OLYMPUS CORPORATION,目录号:0M04213)
明场显微镜(Primovert,Ziess,目录号:3842010199)
 
软件
 
图像J – Open Comet(Benjamin M. Gyori 和 Gireedhar Venkatachalam,www.cometbio.org)
Excel 2016(微软公司)
GraphPad Prism(GraphPad Software, Inc.,www.graphpad.com)
 
程序
 
A.细胞的制备      
安乐死一个C57BL6 / J小鼠,解剖出后肢和去除多余的组织从骨头分离的股骨和胫骨。
冲洗骨髓细胞置于50 -毫升聚丙烯管中,使用高压灭菌的1 × PBS和26用1号针-毫升注射器,直到骨骼被清除内部。
混合细胞通过使单细胞悬浮液和应变一个40 - μm尼龙过滤器。在 4°C 下以 600 × g离心5 分钟。
通过加入500裂解红细胞μ升1 × RBC裂解缓冲液(稀释在1 × PBS)以沉淀,并通过加入2毫升1- 2分钟后中和反应× PBS。在 4°C 下以 600 × g离心5 分钟。
重悬在1ml 1细胞沉淀× PBS和计数细胞使用一个ħ emocytometer。
孵育所述细胞与所需的抗体(2 μ升/ 5百万个细胞用于林APC和1 μ升/ 5百万个细胞为的c-Kit PE和的Sca-1 PerCP-Cy5.5的)染色ħ ematopoietic祖细胞(APC林鸡尾酒、c-Kit PE、Sca-1 PerCP-Cy5.5) 30 分钟。
在 4°C 下以 600 × g离心5 分钟。洗用1细胞× PBS,并转移到一个5 -毫升FACS管中用于分选。
排序的纯LSK(谱系-的Sca- 1 +的ckit + )群体成1 × PBS中,并存储在4℃直至进一步使用。
注意:或者,MACS 排序可用于分离纯度较低的 HSC 祖细胞群。
 
B.玻片准备      
称取 1 g 琼脂糖并使用微波炉溶解在 100 ml 1 × PBS 中。
PLA Ç E中的塑料容器内的彗星载玻片在倾斜ED位置(图1A)。移出 1 ml 琼脂糖并均匀涂抹并在室温下固化 10-15 分钟(图 1B)。
新鲜制备0.75%的低-熔化(LM)琼脂糖使用1 ×在100 PBS -毫升烧杯中。用微波炉溶解。
注意:将熔化的 0.75% LM 琼脂糖转移到储存瓶中以供进一步使用。使用前,在盛有热水的烧杯中重新熔化。
允许热LM琼脂糖到在37℃下冷却至少20分钟。
以 1:10 (v/v) 的比例将 1 × 10 5 个细胞/ml添加到熔化的 LM 琼脂糖中。
注意:如果您取 500 µl 熔融 LM 琼脂糖,则以1 × 10 5细胞/ml 的浓度加入 50 µl 细胞在 1 × PBS(不含Ca 2+和 Mg 2+ )中。
立即将 50 µl 悬浮液移至彗星载玻片上。使用移液器的尖端将混合物均匀地涂抹在表面区域上并使其凝固(图 1C)。
注意:如果样品混合物在载玻片上分散不均匀,在悬浮细胞前将LM琼脂糖加热至 37°C ,然后重新制备样品混合物。
保持载玻片在4℃下在黑暗中10分钟(例如,发生在一个冰箱)。
注意:井的边缘出现一个清晰的 0.5 毫米环(图 1D)。为了提高样品的粘附于滑动,所述胶凝时间和湿度应该增加。
 
 
图1的制备交流OMET滑动。A. 将干净的Trevigen ® CometSlide TM HT 放入塑料容器内。B. 彗星滑梯预先涂有 1%的琼脂糖。这可确保细胞有效地粘附在载玻片上。用琼脂糖预涂后,让凝胶凝固。C. 0.75%琼脂糖LM含有的细胞中加入逐渐的幻灯片。使凝胶在 4°CD 凝固。凝胶凝固后的彗星滑动。
 
C.裂解和电泳      
制备溶胞溶液(见配方)和PLAC Ë在冰上在使用前至少20分钟。
将所需量的预冷裂解液倒入托盘中并浸入载玻片。将托盘放在冰上或 4°C下 1 小时。
从裂解溶液中取出载玻片并排出多余的。
将载玻片放入 50 ml 预冷的 1 ×中性电泳缓冲液(参见配方)中,在 4°C 下洗涤30 分钟。
倾950ml的预冷的1 ×在中性电泳缓冲液,以将彗星试验® ES罐。放置在电泳滑动托盘和盖与所述幻灯片的滑动托盘重叠(图2A)。
设置的电源至21伏,并且执行electrophores是在4℃下1个小时。
对于其它电泳单元,对齐的载玻片从等距离的电极,加1 ×中性电泳缓冲液不超过荷兰国际集团的滑动(图2B)在0.5以上厘米,施加电压30分钟,在1伏每厘米,测量之间的距离的2电泳槽的相对电极(例如,如果距离为 21 cm,则施加 21 V)。
在冷藏室或冰盘上运行电泳装置,冰盘上有足够的冰来部分覆盖装置(图 2C)。
 
 
图 2. 中性条件下的电泳。A. 裂解后,将彗星载玻片置于电泳装置中,并在缓冲罐中加入中性电泳缓冲液。B. 中性电泳缓冲液不应超过载玻片上方 0.5 cm。C.在 4°C 下以21 伏电压进行电泳1 小时。
 
D.载玻片的染色和可视化      
排出多余的中性电泳缓冲液,并在室温下将载玻片浸入DNA 沉淀溶液中 30 分钟(参见食谱)。
取出载玻片,在室温下将它们浸入 70% 乙醇中 5 分钟。
允许样品至干燥10-1 5分钟在室温下。
注:干燥样品将允许细胞沉降在单个平面内,MAK荷兰国际集团的可视化,记录,和孔定量吨通货膨胀更容易。样品可以在评分前与干燥剂一起在室温下储存。
将 100 µl 稀释的 SYBR ® Gold添加到每个样品中并孵育30 分钟。
注意:Trevigen 提供专为彗星染色而设计的 CometAssay ® Silver Staining Kit。银染色可以帮助在所述彗星上的任何传输光显微镜可视化和永久染色的样本的长期存储。建议样品的前银染色干燥。
轻轻敲击载玻片以去除多余的 SYBR ® Gold 溶液。允许滑动到在黑暗中在室温下完全干燥。
查看使用荧光显微镜的幻灯片,并获得公正的图片的每一个细胞,可能是visuali ž版。
注意:该方法的效率可以通过细胞与基因毒性应激作为待测试一个阳性对照。这里,从年轻的成年人(8周造血干细胞和祖细胞小号旧)(图3A)用来自老年小鼠细胞相比,(2年小号旧)(图3B)。老年小鼠细胞中彗尾的形成清晰可见。两个小区之间的比较类型的基因组DNA中的头部相对于比例的尾可以用作为一个可靠的孔定量吨的通货膨胀的DNA损伤的程度。
 
 
图 3.细胞的染色和可视化。A. 来自幼鼠的 HSC –细胞内的细胞核是完整的,表明没有 DNA 损伤。B.从大的小鼠HSC -在细胞细胞核是不完整的,并且形成头部和彗尾,指示存在DNA损伤。
 
E.计分和一个细胞的nalysis:      
彗星的计分进行中的开放彗星- ImageJ的插件。推出该插件我ÑImageJ的(图4A) 。
通过单击“浏览”按钮(图 4B),将您的图像文件输入到“输入文件”选项。
选择将存储输出文件的输出目录,以及应保存在其中的文件的名称(图 4C 和 4D)。
 
 
图4.彗星小号CORING使用ImageJ 。A.开放的ImageJ软件并点击插件。选择 OpenComet_src_v1.3.1 并单击 OpenComet 。B.一个对话框将打开,在那里你需要浏览文件的输入和输出目录。C. 选择用于评分输入文件的图像。D. 选择输出目录。
 
选择默认分析选项并单击 RUN。
该ANALY ž ED输出的数据将出现在Ø本安输出目录。
的输出文件由输出图像和输出电子表格的。查看所有输出图像以进行错误分析和异常值。请注意为每个图像专门给出的数字(图 5A)。
打开电子表格并手动删除异常值数字s 。
从电子表格中获取 Olive moment 和 Tail moment 的值,并使用 GraphPad PRISM 以图形方式表示数据(图 5B)。
 
 
图5.结果小号的Ç OMET得分。A. 彗头和彗尾的鉴定。B.包含计算出的彗星参数的Excel 电子表格。
 
食谱
 
裂解缓冲液(成分/ 1 ,000毫升)
3.5 M 氯化钠– 146.1 克
100 mM EDTA – 37.2 克
100 mM Trizma 碱– 1.2 克
将成分溶解在 700 ml dH 2 O 中
加入 8 g NaOH 颗粒并使用 HCl 或 NaOH(浓缩)将 pH 值调节至 10 .0
50毫升是需要对每个测定
添加 1% Triton X-100 – 0.5 毫升
10%d imethyl亚砜(DMSO) - 5毫升
1 × Ñ eutral电泳缓冲液
准备 10× Ñ eutral Ë lectrophoresis b uffer:
三b酶(分子量= 121.14。)- 60.57克
钠一cetate(。摩尔重量= 136.08)- 204.12克
溶解在450ml d ħ 2 ö和一个djust的pH值至9.0使用冰醋酸
将体积调节至 500 毫升并过滤除菌。在室温下储存。
在 dH 2 O中将 10 ×原液稀释至 1 ×以制备 1 L工作强度缓冲液并在 4°C 下预冷
DNA沉淀溶液
制备的7.5M的10ml的储备溶液一mmonium一个cetate:NH 4 (。摩尔重量= 77.08)AC = 5.78克
d H 2 O (NH 4 Ac 溶解后)– 10 ml
对于 50 ml DNA 沉淀溶液:
7.5 M NH 4 Ac (mol. wt. = 77.08) – 6.7 ml
95% 乙醇(试剂级)– 43.3 毫升
SYBR ®金染色液
来自 SYBR ® Gold 精矿(10,000 ×在 DMSO 中)
SYBR ® Gold – 1 µl
电泳缓冲液 – 10 ml
4°C避光保存
1 × p磷酸盐-缓冲盐水 (PBS)
制备 10 × PBS 储备液:
氯化钠 – 80 克
氯化钾 – 2 克
Na 2 HPO 4 – 14.4 克
KH 2 PO 4 – 2.4 克
取 800 ml Milli-Q 水,溶解盐,调节 pH 至 7.4 ,定容至 1,000 ml
在 dH 2 O中将 10 ×股票稀释至 1 ×
 
致谢
 
这项工作是由威康信托/ DBT印度联盟奖学金支持(IA / I / 15 /502061分之2)从授予SK和校内资金的科学教育和研究特里凡得琅(IISER TVM)的印度理工学院。在我nstitutional动物设施由资金从科学技术部,印度政府的支持(下FIST方案; SR / FST / LS-II /二百一十七分之二千○十八)。IMR是通过支持一个由高级研究奖学金的大学拨款委员会(UGC),印度。该协议取自 Biswas等人的文章。(2020)“所述的骨膜素/整合素AV轴调控对造血的大小干细胞库在胎儿肝脏” ,并从被修改的协议描述中的彗星Trevigen公司测定试剂盒(比斯瓦斯等人,2020) 。
 
利益争夺
 
作者没有与本研究相关的竞争性财务或非财务利益。
 
伦理
 
用于分离 HSC 以进行 DNA 损伤分析 (C57BL/6J-CD45.2) 的动物在 IISER Thiruvananthapuram 的动物设施中饲养和饲养。根据印度政府环境和森林部 (MoEF) 动物实验控制和监督委员会 (CPCSEA) 提供的指导方针饲养动物。在实验中,小鼠保持在隔离笼中,并允许进入到灭菌酸化水和辐射食物随意。所有动物实验均获得该研究所动物伦理委员会 (IAEC) 的批准。
 
参考
 
1. Beedanagari, S. (2017)。4.11-遗传毒理学。我Ñ :Chackalamannil ,小号。,罗特拉,D 。和沃德,S 。E.(E ds.)。综合药物化学 III .第 195-203 页。爱思唯尔,牛津。      
2. Beerman, I. (2017)。老化的造血干细胞室中 DNA 损伤的积累。精血血液学54(1): 12-18。                    
3. Beerman, I.、Seita, J.、Inlay, MA、Weissman, IL 和 Rossi, DJ (2014)。静止的造血干细胞在衰老过程中积累 DNA 损伤,进入细胞周期后修复。细胞 干细胞15(1):37-50。                    
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引用:Roy, I. M., Nadar, P. S. and Khurana, S. (2021). Neutral Comet Assay to Detect and Quantitate DNA Double-Strand Breaks in Hematopoietic Stem Cells. Bio-protocol 11(16): e4130. DOI: 10.21769/BioProtoc.4130.
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