搜索

Whole-mount Enteroid Proliferation Staining
对小肠培养物进行EdU整染和增殖分析   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Small intestinal organoids, otherwise known as enteroids, have become an increasingly utilized model for intestinal biology in vitro as they recapitulate the various epithelial cells within the intestinal crypt (Mahe et al., 2013; Sato et al., 2009). Assessment of growth dynamics within these cultures is an important step to understanding how alterations in gene expression, treatment with protective and toxic agents, and genetic mutations alter properties essential for crypt growth and survival as well as the stem cell properties of the individual cells within the crypt. This protocol describes a method of visualization of proliferating cells within the crypt in three dimensions (Barrett et al., 2015). Whole-mount proliferation staining of enteroids using EdU incorporation enables the researcher to view all proliferating cells within the enteroid as opposed to obtaining growth information in thin slices as would be seen with embedding and sectioning, ensuring a true representation of proliferation from the stem cell compartment to the terminally differentiated cells of the crypt.

Materials and Reagents

  1. 12-well MatTek plate with 1.5 coverslip thickness (MATTEK, catalog number: P12G-1.5-14-F )
  2. 18 G non-flexible stainless steel oral gavage needle (Cadence, Inc., catalog number: 7906 )
  3. 10 ml syringe (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: 14-823-2A )
  4. 50 ml sterile Falcon tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339653 )
  5. Sterile 12-well cell culture dish (Sigma-Aldrich, catalog number: CLS3513 )
  6. 15 ml sterile Falcon tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339651 )
  7. Pre-chilled sterile pipette tips
    Notes:
    1. Any tips that are sterile and can be used with the lab’s pipettors.
    2. These tips can be placed in a -20 °C freezer for 30 min or in a 4 °C refrigerator one hour prior to plating.
  8. 70 μM cell strainer (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: 08-771-2 )
  9. C57BL/6 mouse (male or female, 6-8 weeks of age)
  10. Isofluorane (Allivet, catalog number: 50562 )
  11. Ice cold sterile 1x DPBS without calcium or magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-235 )
  12. UltraPure EDTA (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15576-028 )
  13. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  14. D-Sorbitol (Sigma-Aldrich, catalog number: S1876 )
  15. Matrigel® basement membrane matrix (Corning, catalog number: 356237 )
    Note: Thaw at 4 °C the night before use.
  16. Epidermal growth factor (EGF) (R&D Systems, catalog number: 2028-EG-200 )
  17. Noggin (R&D Systems, catalog number: 1967-NG-025/CF )
  18. R-spondin (R&D Systems, catalog number: 3474-RS-050 )
  19. Wnt3A (R&D Systems, catalog number: 1324-WN-010 )
  20. Advanced DMEM/F12 (Thermo Fisher Scientific, GibcoTM, catalog number: 12634-010 )
  21. L-glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-081 )
  22. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-148 )
  23. 1 M HEPES (pH 7.0-7.6, Sterile filtered) (Sigma-Aldrich, catalog number: H0887 )
  24. N2 supplement (R&D Systems, catalog number: AR003 )
  25. B27 supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 17504-044 )
  26. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437-028 )
  27. Click-iT EdU cell proliferation assay (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C10337 )
  28. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  29. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A8531 )
  30. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  31. TO-PRO-3 Iodide (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: T3605 )
  32. Chelation buffer, prepare fresh (see Recipes)
  33. Shaking buffer (see Recipes)
  34. Minigut culture media (see Recipes)
  35. 2% Formaldehyde (see Recipes)

Equipment

  1. Scissors for dissection (Fisher Scientific, catalog number: 08-951-20 )
  2. Sorvall Legend X1R Centrifuge (or other large refrigerated centrifuge)
  3. 37 °C, 5% CO2 cell culture incubator (Thermo Fisher Scientific, model: HERAcell 150i )
  4. LSM 510 META Inverted (or other inverted laser scanning) confocal microscope with 647 (TO-PRO-3) and 488 (EdU) emission filters

Software

  1. ImageJ Image Analysis Software

Procedure

  1. Harvest and culturing of small intestinal enteroids
    1. Anesthetize and sacrifice mouse with isofluorane followed by cervical dislocation.
    2. Dissect out approximately 10 cm of the proximal intestine (duodenum, near stomach, Figure 1A).
    3. Fill a 10 ml syringe with ice cold PBS and attach the oral gavage needle to the syringe.
    4. Insert the gavage needle into the proximal intestine (from step A2). Flush the intestine with ice cold PBS (Figure 1B).
    5. Cut the intestine open lengthwise (Figure 1C) and cut into 1 cm pieces and transfer into 5 ml ice cold PBS in a 15 ml conical tube (Figure 1D). Vortex for 3 sec to remove loose villi.


      Figure 1. Processing of the small intestine for crypt isolation. A. After removal of the small intestine, the most proximal 10 cm should be collected for further processing. B. Then this section should be flushed with ice cold PBS using the gavage needle attached to the 10 ml syringe. C. The intestinal section is then bisected. D. The bisected intestine is then cut into approximately 1 cm sections and is transferred to a 15 ml conical tube containing 5 ml ice cold PBS.

    6. Pipette off PBS supernatant and discard.
    7. Wash the remaining intestinal pieces with 10 ml ice cold PBS, vortex, and remove supernatant with a pipettor and discard.
    8. Transfer tissue to 5 ml chelation buffer and rock at 4 °C for 10 min.
    9. Remove chelation buffer from the tissue with a pipettor and wash tissue twice with 10 ml ice cold PBS.
    10. Add 5 ml ice cold PBS.
    11. Shake for 2 min. Do not shake with too much vigor. The shake involves full arm motion from the elbow at an even tempo (one down stroke every second, Video 1).

      Video 1. Crypt isolation example

    12. Pour off supernatant from first shake and discard because this supernatant will primarily contain villi.
    13. Add 5 ml PBS and repeat shake for 2 min. Check the supernatant for crypts. To initially check crypts, pipette a 20 μl drop of the PBS supernatant onto a well of a 12 well cell culture dish and view at 4x magnification under a cell culture scope. Look for multiple crypts in which the dark granularity of the Paneth cells can be seen (Figure 2). After this shake, the supernatant should primarily contain villi and look like the image on the left of Figure 2.


      Figure 2. Representative images of villi and crypts at 4x and 20x magnification. Red arrow heads indicate crypts. The images on the left are representative of those likely seen after step A13. The images on the right demonstrate the optimal crypt composition which will be seen after step A18.

    14. Usually, crypts are not seen here and the supernatant can be poured off as well, but this is very dependent on shaking technique so the supernatant should be checked for crypts.
    15. Add 5 ml fresh chelation buffer and chelate for 10 min at 4 °C on the rocker.
    16. Decant the chelation buffer, being careful not to lose the 1 cm pieces of small intestine then add 5 ml ice cold PBS (wash). Discard the PBS and perform a second wash. Discard the wash and add 5 ml ice cold PBS for shaking.
    17. Shake for 2 min. Check for crypts. If you have them here, you can filter and save, but usually the best crypts are seen after a second 2-min shake. Though results will differ based on shaking variability, the supernatant from this shake will usually contain 90% villi and 10% or less crypts.
    18. Perform a second 2-min shake and check for crypts. After this second shake, there is typically an equivalent number of crypts and villi. Ultimately, a ratio of greater than or equal to 70% crypts to 30% villi is optimal. If crypts are still not seen in abundance, shake more vigorously for 30 sec intervals and continue to check for crypts until at least 20 crypts per 20 μl aliquot are seen.
    19. Filter through a 70 μm filter into a 50 ml tube. Rinse filter with 5 ml cold shaking buffer.
    20. Crypts are clearly visible at 4x magnification and can be counted within a representative 20 μl crypt aliquot. At least 20 crypts per 20 μl of the solution should be expected, resulting in a typical total yield of approximately 5,000 crypts. Count crypts and transfer enough volume for 1,500 crypts per two wells to pre-chilled 15 ml conical tubes.
    21. Centrifuge at 1,000 rpm for 10 min at 4 °C. Aspirate off shaking buffer being careful not to disturb the pellet.
    22. Using pre-chilled pipette tips and maintaining all reagents on ice, resuspend crypts in 50 μl of Matrigel per well, containing:
      1. EGF: 50 ng/ml (0.25 μl of 100 μg/ml per 50 μl of Matrigel)
      2. Noggin: 100 ng/ml (1 μl of 50 mg/ml per 50 μl of Matrigel)
      3. R-Spondin: 500 ng/ml (1 μl of 250 μg/ml per 50 μl of Matrigel)
      4. For initial cultures, can also spike with Wnt3a (2 μl 50 μg/ml Wnt3a per 50 μl of Matrigel). This step is not necessary but will increase the plating efficiency of the enteroids.
      Notes:
      a. Store growth factors in very small aliquots at -80 °C.
      b. When resuspending crypts, be sure to resuspend completely without adding bubbles. Pipette in and out using a P200 set for a volume 25 μl less than the total volume and never fully expel or pull in the Matrigel/crypt mixture. This step can be performed at room temperature in a cell culture hood, keeping reagents in an ice bucket until plating.
    23. Using pre-chilled pipette tips, plate onto the coverslip portion of the MatTek plate so that the Matrigel forms a mounded dome in the center of the well (Figure 3A).
    24. Place plate in incubator at 37 °C for 30 min (or longer, up to 2 h) to allow Matrigel to polymerize fully.
    25. Overlay Matrigel with 350 μl Minigut culture media (Figure 3B).


      Figure 3. Images of matrigel plating onto 12-well MatTek plates. A. Matrigel containing crypts is pipetted into the center of the well, avoiding any bubble formation. B. After the Matrigel has set, media is added to each well.

  2. Proliferation staining
    1. Proliferation staining can be conducted at any point post-enteroid plating, depending on the process to be analyzed. If initial growth rates are in question, enteroids can be stained after only a day in culture. If response to treatments is to be ascertained, it is best to allow the enteroids to grow for at least 4 days prior to proliferation staining to ensure that they have formed full crypts (see Figure 4 for comparisons of enteroids at different times post-plating). Treatment can also be performed at a specified time prior to staining, depending on the desired output.


      Figure 4. Representative images of enteroids 1 day, 3 days, and 5 days post-plating

    2. Proliferation is determined using the Click-iT EdU cell proliferation assay according to the manufacturer’s instructions as follows.
    3. Enteroid media is replaced with fresh pre-warmed Minigut media and a 10 μM working solution of EdU.
    4. Incubate enteroids for 15 min under normal growth conditions.
    5. Remove media from enteroids and replace with 1 ml of 2% formaldehyde in PBS and incubate overnight at 4 °C.
    6. The next morning, gently remove the fixative and wash the enteroids twice with 1 ml of 3% BSA in PBS.
    7. Gently remove the last wash and add 1 ml of 0.5% Triton X-100 in PBS and incubate at room temperature for 20 min.
    8. Prepare the Click-iT reaction cocktail (according to manufacturer’s instructions) and add 500 μl per well. Rock the plate briefly to ensure even distribution. Incubate the enteroids for 30 min at room temperature, protected from light.
    9. Remove the reaction cocktail and wash each well once with 1 ml 3% BSA in PBS.
    10. Remove wash solution and counterstain nuclei with 500 μl of TO-PRO-3 (1:500 in PBS) for 15 min at room temperature. TO-PRO-3 enables both non-proliferating and proliferating cells to be identified by their nuclei so that proliferation percentages can be determined.

  3. Imaging of small intestinal enteroids
    1. Enteroids can be imaged on an LSM 510 META or similar inverted confocal microscope. Imaging is performed in the 488 (EdU) and 647 (TO-PRO-3) channels and Z-stacks are taken to ensure three-dimensional imaging of the enteroid (Figure 5A-B).
    2. Proliferation can be quantified as EdU+ cells per crypt area where crypt area is determined using ImageJ image analysis software. Alternatively, proliferation can be quantified as percentage of EdU+ cells relative to total cell number as determined by TO-PRO-3+ nuclei.


      Figure 5. Representative images of proliferating enteroids. Enteroids have been stained for the proliferation marker EdU (green) at (A) 3 days and (B) 5 days post-plating. Counterstain is TO-PRO-3 (nuclei, red).

Notes

  1. It is essential to differentiate between crypts and villi (Figure 1). Become comfortable with detecting crypts and determine the optimal shaking protocol for maximal crypt isolation to ensure optimal plating efficiency. Villi are commonly separated from the mucosa in earlier shakes and crypts in later shakes. Perform experiments to increase reproducibility in your hands.
  2. Ensure that the chelation buffer does not contain calcium (PBS without additional calcium or magnesium) as calcium will disrupt the chelation procedure.

Recipes

  1. Chelation buffer (prepare fresh)
    1 mM EDTA
    PBS
    Note: Make sure PBS is correct pH. Best to use the purchased 1x DPBS without calcium or magnesium for all solutions.
  2. Shaking buffer
    43.3 mM sucrose
    54.9 mM sorbitol
    PBS
    Can be stored at 4 °C
  3. Minigut culture media
    Advanced DMEM/F12
    2 mM L-glutamine
    200 U/ml penicillin
    200 μg/ml streptomycin
    Freeze this in 43.5 ml aliquots and, upon thawing, add:
    20 mM HEPES
    1x N2 supplement
    1x B27 supplement
    10% FBS
    Note: If growing cultures for longer than 4 days, every 4 days, media should be replaced with fresh, complete Minigut media with the following growth factors:
    1 μg/ml R-spondin 1
    100 ng/ml Noggin
    50 ng/ml EGF
  4. 2% formaldehyde
    2 g paraformaldehyde powder
    100 ml of 1x PBS
    Heat to, but do not exceed, 70 °C in a fume hood until the paraformaldehyde dissolves
    Allow the solution to return to room temperature
    Adjust the pH to 7.4 as needed
    Filter and stored at 4 °C

Acknowledgments

This protocol was first described in Barrett et al. (2015). This work was supported by NIH grants DK080221 (to C. S. Williams), 1F31CA167920 (to C. W. Barrett), T32CA009592-26 (to C. W. Barrett), and Merit Review Grants from the Office of Medical Research, Department of Veterans Affairs, 1I01BX001426 (to C. S. Williams).

References

  1. Barrett, C. W., Reddy, V. K., Short, S. P., Motley, A. K., Lintel, M. K., Bradley, A. M., Freeman, T., Vallance, J., Ning, W., Parang, B., Poindexter, S. V., Fingleton, B., Chen, X., Washington, M. K., Wilson, K. T., Shroyer, N. F., Hill, K. E., Burk, R. F. and Williams, C. S. (2015). Selenoprotein P influences colitis-induced tumorigenesis by mediating stemness and oxidative damage. J Clin Invest 125(7): 2646-2660.
  2. Mahe, M. M., Aihara, E., Schumacher, M. A., Zavros, Y., Montrose, M. H., Helmrath, M. A., Sato, T. and Shroyer, N. F. (2013). Establishment of gastrointestinal epithelial organoids. Curr Protoc Mouse Biol 3(4): 217-240.
  3. Sato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange, D. E., van Es, J. H., Abo, A., Kujala, P., Peters, P. J. and Clevers, H. (2009). Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459(7244): 262-265.

简介

小肠类器官,也称为肠袢,已经成为越来越多地用于肠道生物学的体外模型,因为它们重现了在肠隐窝内的各种上皮细胞(Mahe等人 ,2013; Sato et al。,2009)。在这些培养物中评估生长动力学是理解基因表达的改变,用保护性和毒性试剂处理以及遗传突变改变隐窝生长和存活所必需的性质以及细胞内单个细胞的干细胞性质的重要步骤地穴。该方案描述了在隐窝内三维的增殖细胞的可视化方法(Barrett等人,2015)。使用EdU掺入的肠衣的整体增殖染色使得研究者能够观察到肠内的所有增殖细胞,而不是如在包埋和切片中所见到的在薄切片中获得生长信息,确保来自干细胞区室的增殖的真实表现到隐窝的终末分化细胞。

材料和试剂

  1. 具有1.5盖玻片厚度的12孔MatTek板(MATTEK,目录号:P12G-1.5-14-F)
  2. 18G非柔性不锈钢口腔管饲针(Cadence,Inc.,目录号:7906)
  3. 10ml的注射器(Thermo Fisher Scientific,Fisher Scientific ,目录号:14-823-2A)
  4. 50ml无菌Falcon管(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:339653)
  5. 无菌12孔细胞培养皿(Sigma-Aldrich,目录号:CLS3513)
  6. 15ml无菌Falcon管(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:339651)
  7. 预冷无菌移液器吸头
    注意:
    1. 任何无菌的提示,可以与实验室的移液器一起使用。
    2. 这些尖端可以在电镀之前1小时置于-20℃的冷冻器中30分钟或在4℃的冰箱中。
  8. 70μM细胞过滤器(Thermo Fisher Scientific,Fisher Scientific ,目录号:08-771-2)
  9. C57BL/6小鼠(雄性或雌性,6-8周龄)
  10. 异氟烷(Allivet,目录号:50562)
  11. 无钙或镁的冰冷无菌1x DPBS(Thermo Fisher Scientific,Gibco TM ,目录号:14190-235)
  12. UltraPure EDTA(Thermo Fisher Scientific,Invitrogen TM,目录号:15576-028)
  13. 蔗糖(Sigma-Aldrich,目录号:SO389)
  14. D-山梨醇(Sigma-Aldrich,目录号:S1876)
  15. Matrigel 基底膜基质(Corning,目录号:356237)
    注意:使用前的夜晚在4°C解冻。
  16. 表皮生长因子(EGF)(R& D Systems,目录号:2028-EG-200)
  17. Noggin(R& D Systems,目录号:1967-NG-025/CF)
  18. R-spondin(R& D Systems,目录号:3474-RS-050)
  19. Wnt3A(R& D Systems,目录号:1324-WN-010)
  20. Advanced DMEM/F12(Thermo Fisher Scientific,Gibco TM ,目录号:12634-010)
  21. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM ,目录号:25030-081)
  22. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM ,目录号:15140-148)
  23. 1 M HEPES(pH 7.0-7.6,无菌过滤)(Sigma-Aldrich,目录号:H0887)
  24. N2补充剂(R& D Systems,目录号:AR003)
  25. B27补充物(Thermo Fisher Scientific,Gibco TM ,目录号:17504-044)
  26. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10437-028)
  27. Click-iT EdU细胞增殖测定(Thermo Fisher Scientific,Molecular Probes TM ,目录号:C10337)
  28. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
  29. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A8531)
  30. Triton X-100(Sigma-Aldrich,目录号:X100)
  31. TO-PRO-3碘化物(Thermo Fisher Scientific,Molecular Probes TM ,目录号:T3605)
  32. 螯合缓冲液,准备新鲜(见配方)
  33. 摇晃缓冲区(请参阅配方)
  34. Minigut培养基(参见配方)
  35. 2%甲醛(见配方)

设备

  1. 剪刀(Fisher Scientific,目录号:08-951-20)
  2. Sorvall Legend X1R离心机(或其他大型冷冻离心机)
  3. 37℃,5%CO 2细胞培养箱(Thermo Fisher Scientific,型号:HERAcell 150i)中。
  4. LSM 510 META具有647(TO-PRO-3)和488(EdU)发射滤光片的倒置(或其他倒置激光扫描)共聚焦显微镜

软件

  1. ImageJ图像分析软件

程序

  1. 收获和培养小肠肠道
    1. 麻醉和牺牲鼠标异氟烷,然后颈椎脱臼
    2. 解剖近10厘米的近端肠(十二指肠,附近的胃,图1A)。
    3. 用冰冷的PBS填充10 ml注射器,并将口服管饲针注射到注射器
    4. 将管饲针插入近端肠(从步骤A2)。用冰冷的PBS冲洗肠(图1B)。
    5. 切开肠纵向打开(图1C),切成1厘米的块,并转移到15毫升锥形管(图1D)5ml冰冷PBS。涡旋3秒以去除松散的绒毛

      图1.用于隐窝隔离的小肠的处理。A.移除小肠后,应收集最近的10cm用于进一步处理。 B.然后使用连接到10ml注射器的管饲针将该部分用冰冷的PBS冲洗。然后将肠切片二等分。 D.然后将对分的肠切成约1cm切片,并转移到含有5ml冰冷PBS的15ml锥形管中。

    6. 吸取PBS上清液并丢弃。
    7. 用10ml冰冷的PBS洗涤剩余的肠片,涡旋,并用移液管去除上清液并丢弃。
    8. 转移组织到5毫升螯合缓冲液和岩石在4℃下10分钟
    9. 用移液器从组织中移除螯合缓冲液,用10毫升冰冷的PBS洗涤组织两次。
    10. 加入5 ml冰冷PBS。
    11. 摇动2分钟。 不要用太多的活力摇动。 该震动包括以均匀的速度从肘部全臂运动(每秒一次向下冲击,视频1)

    12. 倒掉第一次摇动的上清液并丢弃,因为这种上清液将主要含有绒毛。
    13. 加入5 ml PBS,重复摇动2分钟。检查上清液的隐窝。要开始检查隐窝,移取20微升的PBS上清液到12孔细胞培养皿的井和视图在4倍放大在细胞培养范围内。寻找多个隐窝,其中可以看到Paneth细胞的黑色颗粒(图2)。摇动后,上清液应主要含有绒毛,看起来像图2左侧的图像。


      图2.绒毛和隐窝在4x和20x放大率下的代表图像。红色箭头表示隐窝。左侧的图像代表在步骤A13之后可能看到的那些图像。右侧的图像显示了在步骤A18之后将看到的最佳隐窝组合物
    14. 通常,这里没有看到隐窝,也可以倒出上清液,但是这非常依赖于摇动技术,因此应检查上清液的隐窝。
    15. 加入5毫升新鲜的螯合缓冲液,并在摇床上4℃螯合10分钟
    16. 滗析螯合缓冲液,小心不要失去1厘米的小肠,然后加入5毫升冰冷的PBS(洗涤)。丢弃PBS并执行第二次洗涤。弃去洗液,加入5 ml冰冷的PBS摇匀
    17. 摇动2分钟。检查隐窝。如果你在这里,你可以过滤和保存,但通常最好的隐窝是在第二个2分钟摇动后看到。虽然结果将根据摇晃变异性而不同,但是这种摇动的上清液通常含有90%绒毛和10%或更少的隐窝。
    18. 执行第二个2分钟摇动并检查隐窝。在第二次摇晃之后,通常存在相等数量的隐窝和绒毛。最终,大于或等于70%隐窝与30%绒毛的比率是最佳的。如果隐窝仍然没有看到丰富,更剧烈摇动30秒间隔,继续检查隐窝,直到看到至少20隐窝每20微升等分试样。
    19. 通过70μM过滤器过滤到50ml管中。用5ml冷摇动缓冲液冲洗过滤器。
    20. 隐窝在4倍放大率下清晰可见,并且可以在代表性的20μl隐窝等分试样中计数。应该预期每20μl溶液至少20个隐窝,产生约5,000个隐窝的典型总产量。计数隐窝,并将足够的体积,每两个孔1500个隐窝转移到预冷的15ml锥形管中。
    21. 在4℃下以1,000rpm离心10分钟。吸出摇动缓冲液,小心不要打扰沉淀
    22. 使用预先冷却的移液器吸头和保持所有的试剂在冰上,重悬在每孔50微升Matrigel的隐窝,包含:
      1. EGF:50ng/ml(0.25μl100μg/ml每50μlMatrigel)
      2. 头蛋白:100ng/ml(1μl50mg/ml每50μlMatrigel)
      3. R-Spondin:500ng/ml(1μl250μg/ml每50μlMatrigel)
      4. 对于初始培养物,也可以用Wnt3a(2μl50μg/ml Wnt3a /50μlMatrigel)刺突。该步骤不是必需的,但是将增加肠类的电镀效率。
      注意:
      a。 将生长因子以非常小的等分试样存储在-80°C。
      b。重新悬浮隐窝时,请务必完全重悬,不要加入气泡。移液器进出使用P200集体积小于总体积25微升,从未完全排除或拉入Matrigel/crypt混合物。该步骤可以在室温下在细胞培养罩中进行,将试剂保持在冰桶中直到铺板。
    23. 使用预冷的移液器吸头,平板到MatTek板的盖玻片部分,使Matrigel在孔中心形成一个隆起的圆顶(图3A)。
    24. 将培养板置于37℃的培养箱中30分钟(或更长,最多2小时),使Matrigel完全聚合。
    25. 用350μlMinigut培养基重叠Matrigel(图3B)

      图3.将Matrigel电镀到12孔MatTek平板上的图像。将含有隐窝的基质胶移液到孔的中心,避免任何气泡形成。 B.在Matrigel设置后,将媒体添加到每个孔中。

    26. 增殖染色
      1. 根据待分析的过程,可以在肠后注入的任何点进行增殖染色。如果最初的生长速率是有问题的,只有一天后,肠道菌群可以在培养中染色。如果要确定对治疗的反应,则最好允许肠内生长在增殖染色前生长至少4天,以确保它们形成完全隐窝(参见图4,用于在涂布后不同时间的肠类似物的比较) 。根据所需的输出量,也可以在染色前的指定时间进行处理

        图4.电镀后1天,3天和5天的代表性图像

      2. 根据制造商的说明书,使用Click-iT EdU细胞增殖测定法测定增殖
      3. 用新鲜的预热的Minigut培养基和10μMEdU的工作溶液替换类肠毒素培养基
      4. 在正常生长条件下孵育肠道15分钟。
      5. 从瓶中取出介质,更换为1毫升的2%甲醛的PBS溶液,在4℃下孵育过夜
      6. 第二天早晨,轻轻取出固定液,用1ml 3%BSA的PBS溶液洗两次
      7. 轻轻取出最后一次洗涤,加入1ml 0.5%Triton X-100的PBS溶液,在室温下孵育20分钟。
      8. 准备Click-iT反应鸡尾酒(根据制造商的说明),并添加每孔500μl。短暂摇动板以确保均匀分布。在室温下孵育肠衣30分钟,避光保护
      9. 取出反应混合物,用1ml含3%BSA的PBS洗涤各孔一次
      10. 用500μl的TO-PRO-3(PBS中1:500)在室温下去除洗涤溶液并复染细胞核15分钟。 TO-PRO-3使得非增殖和增殖细胞能够通过其细胞核鉴定,从而可以确定增殖百分比。

    27. 成像的小肠肠道
      1. 类肠毒素可以在LSM 510 META或类似的反向共聚焦显微镜上成像。在488(EdU)和647(TO-PRO-3)通道中进行成像,并且采取Z堆叠以确保肠道的三维成像(图5A-B)。
      2. 增殖可以量化为每个隐窝区域的EdU + sup +细胞,其中使用ImageJ图像分析软件确定隐窝面积。或者,增殖可以量化为相对于通过TO-PRO-3 + 核确定的总细胞数的EdU + 细胞的百分比。

        图5.增殖性肠道的代表性图像。在铺板后的(A)3天和(B)5天时,对于增殖标记EdU(绿色)已经对类毒素进行染色。复染色为TO-PRO-3(核,红色)
    28. 笔记

      1. 区分隐窝和绒毛是必要的(图1)。 舒适地检测隐窝,并确定最佳的隐窝隔离的最佳摇动协议,以确保最佳电镀效率。 绒毛通常在早期的摇动中与粘膜分离,并在随后的摇动中与隐窝分离。 进行实验以提高手中的重现性。
      2. 确保螯合缓冲液不含钙(不含钙或镁的PBS),因为钙会破坏螯合程序。

      食谱

      1. 螯合缓冲液(新鲜制备)
        1mM EDTA
        PBS
        注意:确保PBS的pH正确。 最好使用购买的1x DPBS,不含钙或镁的所有解决方案。
      2. 摇动缓冲区
        43.3mM蔗糖 54.9mM山梨醇 PBS
        可以在4°C下存储
      3. Minigut培养基
        高级DMEM/F12
        2mM L-谷氨酰胺 200 U/ml青霉素
        200μg/ml链霉素 将其以43.5ml等分试样冷冻,解冻后加入:
        20 mM HEPES
        1x N2补助
        1x B27补充
        10%FBS
        注意:如果生长培养物超过4天,每4天,培养基应该用具有以下生长因子的新鲜,完整的Minigut培养基替换:
        1μg/ml R-spondin 1
        100 ng/ml Noggin
        50 ng/ml EGF
      4. 2%甲醛
        2g多聚甲醛粉末
        100 ml 1x PBS
        在通风橱中加热至70℃,但不超过70℃,直到多聚甲醛溶解
        让溶液恢复到室温
        根据需要将pH调节到7.4 过滤并在4℃下贮存

      致谢

      该方案最早在Barrett等人(2015)中描述。这项工作由NIH授予DK080221(向CS Williams),1F31CA167920(向CW Barrett),T32CA009592-26(向CW Barrett)和Merit Review Grants从医学研究室,退伍军人事务部1I01BX001426(至CS威廉姆斯)。

      参考文献

      1. 1. Barrett,CW,Reddy,VK,Short,SP,Motley,AK,Lintel,MK,Bradley,AM,Freeman,T.Vallance,J.,Ning,W.,Parang,B.,Poindexter, Fingleton,B.,Chen,X.,Washington,MK,Wilson,KT,Shroyer,NF,Hill,KE,Burk,RF and Williams,CS(2015)。  硒蛋白P通过介导干性和氧化损伤影响结肠炎诱导的肿瘤发生。 125(7):2646-2660。
      2. Mahe,MM,Aihara,E.,Schumacher,MA,Zavros,Y.,Montrose,MH,Helmrath,MA,Sato,T.and Shroyer,NF(2013)。  建立胃肠上皮器官。 Curr Protoc Mouse Biol 3 ):217-240。
      3. Sato,T.,Vries,RG,Snippert,HJ,van de Wetering,M.,Barker,N.,Stange,DE,van Es,JH,Abo,A.,Kujala,P.,Peters,PJand Clevers, H.(2009)。  Single Lgr5 stem cells build crypt -villus结构体外 没有间充质细胞。 459(7244):262-265。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Barrett, C. W., Short, S. P., Choksi, Y. A. and Williams, C. S. (2016). Whole-mount Enteroid Proliferation Staining. Bio-protocol 6(12): e1837. DOI: 10.21769/BioProtoc.1837.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。