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Immunostaining of Formaldehyde-fixed Metaphase Chromosome from Untreated and Aphidicolin-treated DT40 Cells
对照和蚜肠酶素处理的DT40细胞中经甲醛固定的中期染色体的免疫染色   

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参见作者原研究论文

本实验方案简略版
The Journal of Cell Biology
Aug 2015

Abstract

During mitosis chromosomes are condensed into dense X-shaped structures that allow for microscopic determination of karyotype as well as inspection of chromosome morphology.

This protocol describes a method to perform immunostaining of formaldehyde-fixed metaphase chromosomes from the avian cell line DT40. It was developed to characterize the localization of YFP-tagged TopBP1 on mitotic chromosomes and specifically determine the percentage of TopBP1 foci that formed on breaks/gaps as well as ends of individual metaphase macrochromosomes (Pedersen et al., 2015). For this purpose immunostaining of YFP was applied. However, the protocol may be optimized for other cell lines or epitopes.

Keywords: Metaphase chromosomes (中期染色体), Cytogenetics (细胞遗传学), Genomic instability (基因组不稳定性), Common fragile sites (普通型脆性位点), Replication stress (复制压力), Immunofluorescence (免疫荧光), Microscopy (显微镜检查)

Background

Microscopic analysis of stained metaphase chromosomes is a classical cytogenetic technique that is extensively used for both research and diagnostics. The basic principle involves induction of cell cycle arrest in metaphase by a spindle destabilizing reagent such as colcemid, which will trigger the spindle assembly checkpoint and therefore arrests cells in metaphase. This serves to enrich for cells with condensed chromosomes. Subsequently cells are subjected to swelling in hypotonic solution followed by spreading of mitotic cells on a microscope slide. The final result is microscopically detectable chromosomes from single cells convenient for karyotype analysis as well as investigations of individual chromosomes. Traditionally, swollen cells are fixed with methanol and acetic acid (3:1) before spreading on slides (Hungerford, 1965; Ronne et al., 1979).

The method described here uses formaldehyde rather than methanol for fixation. This can be useful for subsequent staining with antibodies that are not compatible with methanol fixation. The protocol is optimized for metaphase spreads from chicken DT40 cells, and immunostaining of YFP-tagged TopBP1 on metaphase macrochromosomes (Pedersen et al., 2015). TopBP1 foci on mitotic chromosomes mark DNA insults that are transmitted to G1 daughter cells (Pedersen et al., 2015; Gallina et al., 2016; Oestergaard and Lisby, 2016). The fluorescent signal of YFP is lost during the preparation of metaphase spreads, therefore this protocol includes immunostaining of the YFP epitope. However, it should be possible to apply the protocol for other cell lines and epitopes by optimizing incubation time in hypotonic buffer and antibody concentrations, respectively.

Aphidicolin is a replication inhibitor, which at low concentration induces formation of gaps and breaks on metaphase chromosomes preferentially at common fragile sites (Durkin and Glover, 2007). As stated by this protocol, DT40 cells may be subjected to 0.5 μM aphidicolin to induce breaks and gaps on metaphase chromosomes in DT40 (Pedersen et al., 2015).

The avian karyotype comprises macrochromosomes as well as mini and microchromosomes. The latter two groups are too small to reliably determine features such as breaks/gaps or ends. They are therefore not included in this analysis.

Materials and Reagents

  1. 15 ml tubes (such as Sigma-Aldrich, catalog number: T1943 )
  2. Pipette tips  
  3. Round cover slips (Ø12 mm) (Thermo Fisher Scientific)
  4. Cytospin slides (Thermo Fisher Scientific, catalog number: 5991059 )
  5. A DT40 cell line (Baba and Humphries, 1985) carrying endogenous YFP-tagging at the TopBP1 gene (Germann et al., 2014; Pedersen et al., 2015)
  6. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D4540 )
  7. Aphidicolin (Sigma-Aldrich, catalog number: A0781 )
  8. Colcemid (Thermo Fisher Scientific, GibcoTM, catalog number: 15212012 )
  9. Cytofunnels (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: A78710020 )
  10. Nail polish
  11. RPMI 1640 medium GlutaMAX (Thermo Fisher Scientific, GibcoTM, catalog number: 61870044 )
  12. Chicken serum (such as Sigma-Aldrich, catalog number: C5405 or Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 16110082 )
  13. Fetal bovine serum (FBS) (Heat inactivated) (Thermo Fisher Scientific, GibcoTM, catalog number: 10500 )
  14. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  15. Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  16. Potassium chloride (KCl) (TH GEYER, Chemsolute®, catalog number: 1632 )
  17. Paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
  18. Sodium hydroxide (NaOH)
  19. Hydrochloric acid (HCl)
  20. Sodium chloride (NaCl) (Avantor® Performance Materials, J.T. Baker, catalog number: 0278.1000 )
  21. 2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris, Trizma base) (Sigma-Aldrich, catalog number: T1503 )
  22. (Ethylenedinitrilo)tetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: 27285 )
  23. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  24. Tween-20 (Sigma-Aldrich, catalog number: P9416 )
  25. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A4503 )
  26. Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific, Invitrogen, catalog number: A21121 )
  27. Mouse anti-GFP antibody (Roche Diagnostics, catalog number: 11814460001 )
  28. Glycerol (AppliChem, catalog number: 142329.1211 )
  29. n-propyl-gallate (Sigma-Aldrich, catalog number: 02370 )
  30. 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, catalog number: D9542 )
  31. DT40 cells medium (see Recipes)
  32. Aphidicolin solution (see Recipes)
  33. Hypotonic buffer (see Recipes)
  34. 3% paraformaldehyde in PBS (see Recipes)
  35. KCM buffer (see Recipes)
  36. Phosphate buffered saline (PBS) (see Recipes)
  37. PBS-T (see Recipes)
  38. Blocking solution (see Recipes)
  39. Alexa 488-secondary antibody working solution (see Recipes)
  40. Anti-GFP antibody working solution (see Recipes)
  41. DAPI-mounting buffer (see Recipes)

Equipment

  1. Cytocentrifuge, Cytospin4 (Thermo Fisher Scientific, Thermo ScientificTM, model: CytospinTM 4 Cytocentrifuge )
  2. Pipettes (P1000, P200, P20)
  3. CO2 incubator (such as Nuaire, model: 4750E , series 6)
  4. Flasks such as 25 cm2 flasks (TPP, catalog number: 90025 )
  5. Vortex
  6. Fume hood
  7. Coplin jar such as ‘jar staining acc to Coplin W/cover’ (VWR, catalog number: 631-9331 )
  8. Fluorescence microscope (such as GE Healthcare, DeltaVision Elite; Applied Precision) equipped with a 100x objective lens (NA 1.4; Olympus, model: U-PLAN S-APO ), a cooled EM CCD camera (Photometrics, model: Evolve 512 ), and a solid-state illumination source (Insight; Applied Precision) or similar fluorescence microscope. The microscope should be equipped with appropriate filters to image Alexa488 and DAPI

Software

  1. SoftWoRx (Applied Precision) software or similar
  2. Volocity software (PerkinElmer) or ImageJ

Procedure

  1. Day 1
    1. Prepare the following two cell cultures
      1. 5 ml DT40 (TopBP1YFP/+/+) cell culture with a cell density of 0.7 million per ml  DT40 cells medium (RPMI 1640 medium GlutaMAX supplemented with 2% chicken serum, 8% FBS, 55 µM β-mercaptoethanol, 50 U/ml penicillin and 50 µg/ml streptomycin) + DMSO (solvent control, diluted similar to aphidicolin).
      2. 5 ml DT40 (TopBP1YFP/+/+) cell culture with a cell density of 0.7 million per ml DT40 cells medium + 0.5 µM aphidicolin (aphidicolin from 4 mM stock is diluted in culture medium).
    2. Incubate for 16 h at 39 °C in a CO2 incubator with 5% CO2.

  2. Day 2
    1. Add colcemid to 0.1 µg/ml final concentration to the 5 ml cell culture in each of the flasks and incubate for 150 min in the CO2 incubator.
    2. Harvest the culture with 5 ml of colcemid-treated cells by centrifugation for 5 min at 201 x g in 15 ml tubes.
    3. Discard the supernatants and gently vortex the cell pellet to resuspend it in the remaining liquid.
    4. Vortex the tube at very low intensity and add drop by drop 1 ml ice cold hypotonic buffer.
    5. Add drop by drop another 1 ml ice cold hypotonic buffer (no need to vortex).
    6. Incubate for 15 min on ice.
    7. Meanwhile label cytospin slides and carefully place them in the cytofunnel according to manufacturers’ protocol. See Figure 1 for image of the cytofunnel.


      Figure 1. Image showing the cytofunnel. Top arrow indicates the site of loading into the funnel. Bottom arrow indicates the flow direction of the cell suspension toward the slide, which is indicated as a blue line. This takes place during centrifugation with the result that cells are spun onto the slide at the site of the red X.

    8. After the 15 min incubation, transfer 400 μl of the cell suspension from step B5 to the cytofunnel and spin it in a cytospin centrifuge for 5 min at 1,000 rpm corresponding to 113 x g.
    9. Take the slide out of the cytofunnel.
    10. To fix the sample on the slide, carefully add 100 μl paraformaldehyde solution to the marked area on the slide and leave for 10 min in the fume hood (the paraformaldehyde should stay as a droplet on the slide).
    11. Immerse slides in a Coplin jar with 35 ml KCM buffer for 10 min.
    12. Pour out the KCM buffer (hold the slides in place) and then add 35 ml of PBS to the Coplin jar with the slides.
    13. Pour out the PBS (hold the slides in place) and then add 35 ml of PBS to the Coplin jar with the slides once again.
      Note: At this stage you may store the Coplin jar with slides in PBS in the fridge until next day.
    14. After at least 5 min, take out the slides and drain them well.
      Note: Slides should be dry, except from the area within the circle.
    15. Add 30 μl blocking solution to the area within the circle and cover with coverslip.
    16. Leave for 30 min at room temperature and then very carefully remove the coverslip. (Most of the blocking solution is removed along with the coverslip.)
    17. Add 20 μl primary anti-GFP antibody solution to the area within the circle, and cover with a coverslip.
    18. Incubate in a humidified chamber at 37 °C for 25 min.
    19. Remove the coverslip carefully and wash 2 times, for 3 min each time in PBS-T in a Coplin jar.
    20. Drain (slides should be dry, except from the area within the circle) and add 20 μl Alexa-488 secondary antibody working solution to the area within the circle. Then cover the area within the circle with a coverslip.
    21. Incubate in a humidified chamber at 37 °C for 30 min (protected from light).
    22. Remove coverslip carefully and wash 2 times for 3 min each time in PBS-T (35 ml) in a Coplin jar.
    23. Drain (slides should be dry, except from the area within the circle) and mount round coverslip onto glass slide with 6 μl DAPI-mounting buffer.
    24. Seal with a thin layer of nail polish at the edge of the coverslip. See Figure 2 for image of the slide.


      Figure 2. Image of the cytospin slide. The sample is within the white circle. A round coverslip covers the area within the circle and nail polish is used to seal the coverslip along the edges/the white circle.

    25. When the nail polish is dry, store the slides in a slide storage box in the fridge or proceed directly to microscopic analyses of the slides.
      Notes:
      1. Fluorophores may be visualized under a wide-field microscope (DeltaVision Elite; Applied Precision) equipped with a 100x objective lens (U-PLAN S-APO, NA 1.4; Olympus), a cooled EM CCD camera (Evolve 512; Photometrics), and a solid-state illumination source (Insight; Applied Precision) or similar fluorescence microscope.
      2. Images can be acquired using softWoRx (Applied Precision) software or similar.

Data analysis

Images can be processed and analyzed with Volocity software (PerkinElmer) or ImageJ. Macrochromosomes suitable for analysis are selected based on the following criteria; they should be in focus, they should not overlie other chromosomes and they should be longer than 2 μm. Selected macrochromosomes is then analyzed for gaps or breaks and TopBP1 foci localization (Figure 3). Gaps and breaks are defined as areas on chromosomes with no DAPI staining. Many macrochromosomes do not hold TopBP1 foci. When TopBP1 foci are present they can be classified into three categories: I: at chromosome ends, II: at gaps/breaks or III: at internal sites (which corresponds to the remaining foci found on the chromosomes).


Figure 3. Representative images of slides prepared according to the protocol. Images showing interphase nuclei and metaphase chromosome spreads. Macrochromosomes suitable for analysis are encircled by read dashed line. Left panel: metaphase spreads from untr. cells. Top, DAPI staining. Middle, Alexa-488 staining of YFP-tagged TopBP1. Bottom, merge. Right panel: metaphase spreads from aphidicolin-treated cells. Left, DAPI staining. Middle, Alexa-488 staining of YFP-tagged TopBP1. Bottom, merge. Chromosomes with TopBP1 at the end are marked by red asterisks. Chromosome with TopBP1 at a break is marked by a yellow asterisk. Scale bars = 8 μm.

The data can be quantified by analyzing 250-300 macrochromosomes from both untreated and aphidicolin-treated cells from three independent experiments (80-100 from each condition of each experiment). Data can be presented in a column diagram showing for each condition: a. the percentage of macrochromosomes with TopBP1 at the end, b. the percentage of macrochromosomes with TopBP1 at gaps and breaks, and c. the percentage of macrochromosomes with TopBP1 at an internal site. It is recommended that data be analyzed blinded.

Notes

  1. An empty tip box with water in the reservoir can be used as a humidified chamber.
  2. Experiments should be analyzed blinded to avoid bias in quantification.
  3. Optimization of incubation time and temperature (step B6, day 2: incubation in hypotonic buffer) was crucial to obtain good quality metaphase spreads.

Recipes

  1. DT40 cells medium
    RPMI 1640 medium GlutaMAX supplemented with:
    2% chicken serum
    8% FBS
    55 µM β-mercaptoethanol
    50 U/ml penicillin and 50 µg/ml streptomycin
  2. Aphidicolin solution
    Dissolved in DMSO to a stock concentration of 4 mM and aliquots are stored at -20 °C for maximum 6 months. The aphidicolin solution does not need to be sterilized
  3. Hypotonic buffer
    Prepared fresh by mixing 1 volume FBS, 1 volume 75 mM KCl with 3 volumes of (Milli-Q) H2O
  4. 3% paraformaldehyde in PBS
    Dissolve 0.3 g paraformaldehyde in 7 ml (Milli-Q) H2O, 1 ml 10x PBS, 200 μl NaOH (2 N) by warming the solution to 65 °C
    When the paraformaldehyde is dissolved, the solution is adjusted to pH 7.4 with HCl. Finally the volume is adjusted to 10 ml with (Milli-Q) H2O
    After preparation aliquots of 0.5 ml are stored at -20 °C for maximum 12 months
    Note: Aliquots should not be refrozen or reused after the day of thawing.
  5. KCM buffer
    120 mM KCl
    20 mM NaCl
    10 mM Tris/HCl pH 8.0
    0.5 mM EDTA
    0.1% Triton X-100
    Note: KCM buffer can be stored at room temperature for 3 months.
  6. Phosphate buffered saline (PBS), pH 7.4
    37 mM NaCl
    10 mM phosphate
    2.7 mM KCl
  7. PBS-T
    Prepared by adding 0.1% Tween 20 to PBS
    PBS-T can be kept for one month at 4 °C
  8. Blocking solution
    Prepared by supplementing PBS-T with 3% BSA
    The solution can be stored for 5 days at 4 °C
  9. Alexa 488-secondary antibody working solution
    Alexa Fluor 488-conjugated anti-mouse IgG is diluted 1:1,000 in blocking solution
    Note: The solution should be stored on ice, protected from light and used the same day.
  10. Anti-GFP antibody working solution
    Mouse anti-GFP antibody is diluted 1:1,000 in blocking solution, stored on ice and used the same day
  11. DAPI-mounting buffer
    Contains 85% glycerol and 2.5% n-propyl-gallate with 1.5 µg/ml DAPI
    Stored at -20 °C, protected from light, DAPI-mounting buffer can be stored for 6 months

Acknowledgments

This work was supported by the Villum Foundation. This protocol is adapted from (Jeppesen, 2000).

References

  1. Baba, T. W. and Humphries, E. H. (1985). Formation of a transformed follicle is necessary but not sufficient for development of an avian leukosis virus-induced lymphoma. Proc Natl Acad Sci U S A 82(1): 213-216.
  2. Durkin, S. G. and Glover, T. W. (2007). Chromosome fragile sites. Annu Rev Genet 41: 169-192.
  3. Gallina, I., Christiansen, S. K., Pedersen, R. T., Lisby, M. and Oestergaard, V. H. (2016). TopBP1-mediated DNA processing during mitosis. Cell Cycle 15(2): 176-183.
  4. Germann, S. M., Schramke, V., Pedersen, R. T., Gallina, I., Eckert-Boulet, N., Oestergaard, V. H. and Lisby, M. (2014). TopBP1/Dpb11 binds DNA anaphase bridges to prevent genome instability. J Cell Biol 204(1): 45-59.
  5. Hungerford, D. A. (1965). Leukocytes cultured from small inocula of whole blood and the preparation of metaphase chromosomes by treatment with hypotonic KCl. Stain Technol 40(6): 333-338.
  6. Jeppesen, P. (2000). Immunofluorescence in cytogenetic analysis: method and applications. Genet Mol Biol 23:1003-1014.
  7. Oestergaard, V. H. and Lisby, M. (2016). TopBP1 makes the final call for repair on the verge of cell division. Mol Cell Oncol 3(2): e1093066.
  8. Pedersen, R. T., Kruse, T., Nilsson, J., Oestergaard, V. H. and Lisby, M. (2015). TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells. J Cell Biol 210(4): 565-582.
  9. Ronne, M., Andersen, O. and Erlandsen, M. (1979). Effect of colcemid exposure and methanol acetic acid fixation on human metaphase chromosome structure. Hereditas 90(2): 195-201.

简介

在有丝分裂期间,染色体被浓缩成致密X型结构,允许显微检测染色体核型和检查染色体形态。
该方案描述了从禽细胞系DT40进行甲醛固定的中期染色体的免疫染色的方法。 它被开发用于表征YFP标记的TopBP1在有丝分裂染色体上的定位,并且具体确定形成于断裂/间隙以及单个中期大染色体末端的TopBP1灶的百分比(Pedersen等,2015)。 为此,应用YFP的免疫染色。 然而,该协议可以针对其他细胞系或表位进行优化。
【背景】染色中期染色体的显微镜分析是经典的细胞遗传学技术,广泛用于研究和诊断。基本原理包括通过锭子不稳定试剂如谷氨酸诱导中期细胞周期停滞,这将触发主轴装配检查点,从而阻止细胞在中期。这用于富集浓缩染色体的细胞。随后将细胞在低渗溶液中进行溶胀,然后在显微镜载玻片上铺展有丝分裂细胞。最终结果是来自单细胞的显微镜检测染色体,便于染色体核型分析以及个体染色体的研究。传统上,肿胀的细胞用甲醇和乙酸(3:1)固定,然后铺展在载玻片上(Hungerford,1965; Ronne等,1979)。
这里描述的方法使用甲醛而不是甲醇进行固定。这可以用于随后用与甲醇固定不相容的抗体染色。该方案针对鸡DT40细胞的中期传播进行了优化,YFP标记的TopBP1在中期大染色体上的免疫染色(Pedersen等,2015)。 TopBP1对有丝分裂染色体的关注标记了传递给G1子细胞的DNA损伤(Pedersen等,2015; Gallina等,2016; Oestergaard和Lisby,2016)。在制备中期扩散期间,YFP的荧光信号丢失,因此该方案包括YFP表位的免疫染色。然而,应该可以分别通过优化低渗缓冲液和抗体浓度的孵育时间来应用其他细胞系和表位的方案。
Aphidicolin是一种复制抑制剂,其在低浓度下诱导在中期染色体上形成间隙,并优先在常见的脆性位点(Durkin和Glover,2007)。如本方案所述,DT40细胞可能经受0.5μMAphidicolin诱导DT40中后期染色体上的断裂和间隙(Pedersen等,2015)。
禽核型包括大染色体以及微型和微型染色体。后两组太小,无法可靠地确定诸如断裂/间隙或末端之类的特征。因此,它们不包括在此分析中。

关键字:中期染色体, 细胞遗传学, 基因组不稳定性, 普通型脆性位点, 复制压力, 免疫荧光, 显微镜检查

材料和试剂

  1. (如Sigma-Aldrich,目录号:T1943)
  2. 移液器提示
  3. 圆形盖板(Ø12mm)(Thermo Fisher Scientific)
  4. 细胞旋转幻灯片(Thermo Fisher Scientific,目录号:5991059)
  5. 在TopBP1基因(Germann等人,2014; Pedersen等人,2015)上携带内源性YFP标签的DT40细胞系(Baba和Humphries,1985) br />
  6. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D4540)
  7. Aphidicolin(Sigma-Aldrich,目录号:A0781)
  8. Colcemid(Thermo Fisher Scientific,Gibco TM ,目录号:15212012)
  9. Cytofunnels(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:A78710020)
  10. 指甲油
  11. RPMI 1640培养基GlutaMAX(Thermo Fisher Scientific,Gibco TM,目录号:61870044)
  12. 鸡血清(如Sigma-Aldrich,目录号:C5405或Thermo Fisher Scientific,Thermo Scientific TM,目录号:16110082)
  13. 胎牛血清(FBS)(热灭活)(Thermo Fisher Scientific,Gibco TM,目录号:10500)
  14. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  15. 青霉素/链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  16. 氯化钾(KCl)(TH GEYER,Chemsolute ®,目录号:1632)
  17. 多聚甲醛(Sigma-Aldrich,目录号:158127)
  18. 氢氧化钠(NaOH)
  19. 盐酸(HCl)
  20. 氯化钠(NaCl)(Avantor Performance Materials,J.T.Baker,目录号:0278.1000)
  21. 2-氨基-2-(羟甲基)-1,3-丙二醇(Tris,Trizma碱)(Sigma-Aldrich,目录号:T1503)
  22. (Ethylenedinitrilo)tetraacetic acid(EDTA)(Sigma-Aldrich,目录号:27285)
  23. Triton X-100(Sigma-Aldrich,目录号:X100)
  24. 吐温-20(Sigma-Aldrich,目录号:P9416)
  25. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A4503)
  26. Alexa Fluor 488缀合的抗小鼠IgG(Thermo Fisher Scientific,Invitrogen,目录号:A21121)
  27. 小鼠抗GFP抗体(Roche Diagnostics,目录号:11814460001)
  28. 甘油(AppliChem,目录号:142329.1211)
  29. 没食子酸正丙酯(Sigma-Aldrich,目录号:02370)
  30. 2-(4-脒基苯基)-6-吲哚甲脒二盐酸盐,4',6-二脒基-2-苯基吲哚二盐酸盐(DAPI)(Sigma-Aldrich,目录号:D9542)
  31. DT40细胞培养基(参见食谱)
  32. Aphidicolin溶液(参见食谱)
  33. 低音缓冲(见配方)
  34. PBS中3%多聚甲醛(见配方)
  35. KCM缓冲(见配方)
  36. 磷酸盐缓冲盐水(PBS)(见食谱)
  37. PBS-T(见食谱)
  38. 阻塞解决方案(见配方)
  39. Alexa 488-二抗工作解决方案(参见食谱)
  40. 抗GFP抗体工作液(参见食谱)
  41. DAPI安装缓冲区(见配方)

设备

  1. 细胞离心机,Cytospin4(Thermo Fisher Scientific,Thermo Scientific TM,型号:Cytospin TM细胞离心机)
  2. 移液器(P1000,P200,P20)
  3. CO 2培养箱(如Nuaire型号:4750E,系列6)
  4. 烧瓶如25cm 2烧瓶(TPP,目录号:90025)
  5. 涡流
  6. 通风柜
  7. Coplin罐如"Coplin W/cover"的罐染色(VWR,目录号:631-9331)
  8. 配备100倍物镜(NA 1.4; Olympus,型号:U-PLAN S-APO)的荧光显微镜(例如GE Healthcare,DeltaVision Elite; Applied Precision),冷却的EM CCD相机(Photometrics,型号:Evolve 512)和固态照明源(Insight; Applied Precision)或类似的荧光显微镜。显微镜应配备适当的过滤器以图像Alexa488和DAPI

软件

  1. SoftWoRx(Applied Precision)软件或类似的
  2. Volocity软件(PerkinElmer)或ImageJ

程序

  1. 第1天
    1. 准备以下两种细胞培养物
      1. 每ml DT40细胞培养基(RPMI 1640培养基GlutaMAX,补充2%鸡血清,8%FBS,55μMβ-巯基乙醇,50U)的DT40(TopBP1YFP/+/+)细胞培养物/ml青霉素和50μg/ml链霉素)+ DMSO(溶剂对照,稀释类似于aphidicolin)。
      2. 细胞密度为70万/ml的DT40(TopBP1YFP/+/+)细胞培养物,DT40细胞培养基+0.5μMAphidicolin(4mM原液中的aphidicolin在培养基中稀释)。
    2. 在具有5%CO 2的CO 2培养箱中在39℃下孵育16小时。

  2. 第2天
    1. 将最终浓度为0.1μg/ml的谷氨酸加入到每个烧瓶中的5ml细胞培养物中,并在CO 2培养箱中孵育150分钟。
    2. 通过在15 ml管中以201 x x x离心5分钟,将5ml培养基中的细胞收获培养物。
    3. 弃去上清液,轻轻涡旋细胞沉淀,将其重新悬浮在剩余的液体中
    4. 以非常低的强度涡旋管,并逐滴加入1ml冰冷的低渗缓冲液。
    5. 逐滴加入1 ml冰冷的低渗缓冲液(不需要旋涡)
    6. 在冰上孵育15分钟。
    7. 同时标记细胞分裂素幻灯片,并按照制造商的方案将它们小心地放置在细胞碎片中。细胞通道的图像见图1

      图1.显示细胞溢出物的图像。顶部箭头表示装入漏斗的位置。底部箭头表示细胞悬浮液朝向幻灯片的流动方向,以蓝线表示。这在离心过程中发生,结果是将细胞旋转到红色X位置的载玻片上
    8. 孵育15分钟后,将400μl细胞悬浮液从步骤B5转移到细胞悬液中,并在细胞离心机离心机中旋转5分钟,速度为1000转/分,相当于113×g。
    9. 将幻灯片从细胞通道中取出。
    10. 要将样品固定在载玻片上,请小心地将100μl多聚甲醛溶液加入到载玻片上的标记区域,并在通风橱中放置10分钟(多聚甲醛应作为液滴留在载玻片上)。
    11. 将载玻片浸入带有35毫升KCM缓冲液的平底锅中10分钟
    12. 倒出KCM缓冲液(将载玻片放在适当的位置),然后用幻灯片将35ml PBS加入coplin jar。
    13. 倒出PBS(将载玻片保持在适当的位置),然后再次将35ml PBS加入滑板,
      注意:在这个阶段,您可以将冰箱里的幻灯片储存在冰箱中,直到第二天。
    14. 至少5分钟后,取出幻灯片并排好。
      注意:除圆圈内的区域外,幻灯片应干燥。
    15. 将30μl封闭溶液加入圆圈内,盖上盖玻片。
    16. 在室温下放置30分钟,然后小心地取出盖玻片。 (大多数阻塞解决方案与盖玻片一起移除。)
    17. 在圆圈内加入20μl初级抗GFP抗体抗体溶液,盖上盖玻片
    18. 在加湿室中37℃孵育25分钟。
    19. 仔细取出盖玻片,每次洗涤2次,每次3次,每次在PBS-T中,在平底锅中。
    20. 排水(幻灯片应干燥,除了圆圈内的区域),并将20μlAlexa-488二级抗体工作溶液加入到圆圈内。然后用盖玻片覆盖圈内的区域。
    21. 在37℃的加湿室中孵育30分钟(防止光照)。
    22. 仔细取出盖玻片,每次洗涤2次,每次3次,每次在PBS-T(35 ml)中。
    23. 排水(幻灯片应干燥,除了圆圈内的区域),并将圆形盖玻片安装到载玻片上,并放置6μlDAPI安装缓冲液。
    24. 用盖玻片边缘的一层薄薄的指甲油密封。有关幻灯片的图像,请参见图2

      图2.细胞分裂素载玻片的图像。样品在白色圆圈内。圆形盖玻片覆盖圆圈内的区域,指甲油用于沿着边缘/白色圆圈密封盖玻片。

    25. 当指甲油干燥时,将载玻片存放在冰箱的滑动存储箱中,或直接进行幻灯片的微观分析。
      注意:
      1. 荧光团可以在装备有100x物镜(U-PLAN S-APO,NA 1.4; Olympus)的宽视野显微镜(DeltaVision Elite; Applied Precision),冷却的EM CCD相机(Evolve 512; Photometrics )和固态照明源(Insight; Applied Precision)或类似的荧光显微镜。
      2. 可以使用softWoRx(Applied Precision)软件或类似软件获取图像

数据分析

可以使用Volocity软件(PerkinElmer)或ImageJ对图像进行处理和分析。基于以下标准选择适合分析的大染色体;它们应该是焦点,它们不应该覆盖其他染色体,它们应该长于2μm。然后分析选择的大染色体的间隙或断裂和TopBP1病灶定位(图3)。间隙和断裂被定义为染色体上没有DAPI染色的区域。许多大染色体不含TopBP1病灶。当TopBP1焦点存在时,它们可以分为三类:I:染色体末端,II:间隙/断裂或III:内部位点(对应于染色体上剩余的焦点)。


图3.根据方案制备的载玻片的代表性图像。显示相间核和中期染色体扩散的图像。适用于分析的大染色体被读取虚线包围。左图:中期从未开始传播。细胞。顶部,DAPI染色。中性,YFP标记的TopBP1的Alexa-488染色。底部,合并。右图:来自阿奇抑霉素处理的细胞的中期传播。左,DAPI染色。中性,YFP标记的TopBP1的Alexa-488染色。底部,合并。末端具有TopBP1的染色体上标有红色星号。断头处的TopBP1染色体上标有黄色星号。刻度棒=8μm。

可以通过分析来自三个独立实验(来自每个实验的每个条件的80-100)的来自未处理和aphidicolin处理的细胞的250-300个大染色体来量化数据。数据可以显示在每个条件的列图中:a。 b)最后的TopBP1大染色体的百分比。宏观染色体与TopBP1在间隙和断裂处的百分比,以及c。大部分染色体与TopBP1在内部位点的百分比。建议对数据进行盲法分析。

笔记

  1. 水箱内有一个带有水的空头盒可用作加湿室。
  2. 应对实验进行盲法分析,以避免量化偏差
  3. 培养时间和温度的优化(步骤B6,第2天:在低渗缓冲液中孵育)对获得良好质量的中期传播至关重要。

食谱

  1. DT40细胞培养基
    RPMI 1640培养基GlutaMAX补充:
    2%鸡血清
    8%FBS
    55μMβ-巯基乙醇
    50 U/ml青霉素和50μg/ml链霉素
  2. Aphidicolin溶液
    溶解在DMSO中至4mM的储备浓度,并将等分试样储存在-20℃下最多6个月。 aphidicolin溶液不需要消毒
  3. 低音缓冲区
    通过混合1体积FBS,1体积75mM KCl与3体积的(Milli-Q)H 2 O
    制备新鲜
  4. PBS中的3%多聚甲醛
    将0.3g多聚甲醛溶解在7 ml(Milli-Q)H 2 O,1 ml 10x PBS,200μlNaOH(2N)中,将溶液加热至65°C。 当多聚甲醛溶解时,溶液用HCl调节至pH 7.4。最后将体积用(Milli-Q)H 2 O is is调至10ml 制备0.5ml的等分试样在-20℃下储存最多12个月 注意:解冻之后不得重新冷冻或重新使用等分试样。

  5. KCM缓冲区
    120 mM KCl
    20 mM NaCl
    10 mM Tris/HCl pH 8.0
    0.5 mM EDTA
    0.1%Triton X-100
    注意:KCM缓冲液可以在室温下储存3个月。
  6. 磷酸盐缓冲盐水(PBS),pH 7.4
    37 mM NaCl
    10 mM磷酸盐
    2.7 mM KCl
  7. PBS-T
    通过向PBS中加入0.1%吐温20制备
    PBS-T可以在4℃下保存一个月
  8. 阻塞解决方案
    通过用3%BSA补充PBS-T制备 溶液可以在4°C下储存5天
  9. Alexa 488二次抗体工作解决方案
    Alexa Fluor 488缀合的抗小鼠IgG在阻断溶液中以1:1,000稀释 注意:该解决方案应存放在冰上,避光保存,并在当天使用。
  10. 抗GFP抗体工作液
    小鼠抗GFP抗体在封闭溶液中稀释1:1,000,储存在冰上,并在同一天使用
  11. DAPI安装缓冲区
    含有85%甘油和2.5%没食子酸正丙酯,含有1.5μg/ml DAPI 保存在-20°C,防止光照,DAPI安装缓冲液可以存放6个月

致谢

这项工作得到了Villum基金会的支持。该协议来自(Jeppesen,2000)。

参考

  1. Baba,TW和Humphries,EH(1985)。  形成转化的卵泡是必需的,但不足以用于发展禽白血病病毒诱导的淋巴瘤。 Proc Natl Acad Sci USA 82(1):213-216。
  2. Durkin,SG和Glover,TW(2007)。  染色体脆弱的网站。 Annu Rev Genet 41:169-192。
  3. Gallina,I.,Christiansen,SK,Pedersen,RT,Lisby,M.and Oestergaard,VH(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih .com/tubmed/26701150"target ="_ blank"> TopBP1介导的有丝分裂期DNA处理。细胞周期 15(2):176-183。
  4. Germann,SM,Schramke,V.,Pedersen,RT,Gallina,I.,Eckert-Boulet,N.,Oestergaard,VH和Lisby,M。(2014)。 TopBP1/Dpb11结合DNA后期桥以防止基因组不稳定。 J Cell Biol 204(1):45-59。
  5. Hungerford,DA(1965)。从小接种物培养的白细胞的全血和通过用低渗KCl处理制备中期染色体。 染色技术 40(6):333-338。
  6. Jeppesen,P.(2000)。  细胞遗传学分析中的免疫荧光:方法和应用。 Genet Mol Biol 23:1003-1014。
  7. Oestergaard,VH和Lisby,M。(2016)。 TopBP1在细胞分裂的边缘进行修复的最后一个呼叫。 3(2):e1093066。
  8. Pedersen,RT,Kruse,T.,Nilsson,J.,Oestergaard,VH和Lisby,M。(2015)。 TopBP1是有丝分裂所必需的,以减少对G1子细胞的DNA损伤的传播。细胞生物学 210(4):565 -582。
  9. Ronne,M.,Andersen,O.and Erlandsen,M。(1979)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/437988" 目标="_ blank"> colcemid暴露和甲醇乙酸固定对人中期染色体结构的影响。 90(2):195-201。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Oestergaard, V. H. (2017). Immunostaining of Formaldehyde-fixed Metaphase Chromosome from Untreated and Aphidicolin-treated DT40 Cells. Bio-protocol 7(9): e2259. DOI: 10.21769/BioProtoc.2259.
  2. Pedersen, R. T., Kruse, T., Nilsson, J., Oestergaard, V. H. and Lisby, M. (2015). TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells. J Cell Biol 210(4): 565-582.
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