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Invadopodia Detection and Gelatin Degradation Assay
侵足检测和明胶降解分析   

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Abstract

This protocol is designed to quantify invadopodia formation and activity. Invadopodia are protrusive structures elaborated by cancer cells that mediate cell attachment and remodeling of the extracellular matrix. These structures contribute to the ability of cancer cells to invade and metastasize. In this protocol, both the presence of invadopodia and their activity is simultaneously assessed and quantified by a fluorescent microscopy-based assay.

Keywords: Cancer(癌症), Invasion(入侵), Invadopodia(侵袭性伪足), Metastasis(转移), Gelatin(明胶)

Materials and Reagents

  1. Cells (i.e. SCC61 head and neck carcinoma cells)
  2. Tissue culture media (i.e. DMEM) (Mediatech, catalog number: 10013CV )
  3. Sterile PBS
  4. Trypsin 0.05%/EDTA (Life Technologies, catalog number: 25300054 )
  5. Penicillin/Streptomycin solution (Omega Scientific, catalog number PS-20 )
  6. Ethanol (Decon Labs, catalog number: 2801 )
  7. Forceps Dumont #5 (Fine Science Tools, catalog number: 11252-20 )
  8. 25% Glutaraldehyde (Polysciences, catalog number: 00376-500 )
  9. Sucrose (Fisher Scientific, catalog number: S3500 )
  10. Sodium Borohydride (Fisher Scientific, catalog number: S67825 )
  11. 16% Formaldehyde (Electron Microscopy Sciences, catalog number: 15710 )
  12. Gelatin from pig skin, Oregon green 488 conjugate (Life Technologies, catalog number: G13186 )
  13. Alexa Fluor 568 Phalloidin (Life Technologies, catalog number: A12380 )
  14. Mounting medium, Vectashield with DAPI (Vector Laboratories, catalog number: H-1200 )
  15. Bovine Serum Albumin fraction V (EMD Millipore, catalog number: 2980-1KG )
  16. Triton X-100 (Promega corporation, catalog number: H5141 )
  17. Parafilm (Bemis Company, catalog number: PM996 )

Equipment

  1. Round 18 mm diameter glass coverslips (0.13-0.17 mm thickness) (Carolina Biological Supply Company, catalog number: 633033 )
  2. Microscope glass slides (75 x 25 mm) (Thermo Fisher Scientific, catalog number: 1255015 )
  3. 12-well Microplates (Corning, catalog number: 3513 )
  4. Thermomixer or water bath set at 60 °C
  5. Tissue culture incubator
  6. Hemocytometer (LabSource, catalog number: 0267154 ) http://www.lifetechnologies.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/counting-cells-in-a-hemacytometer.html
  7. Fluorescence Microscope
  8. Vacuum source

Procedure

  1. Preparing fluorescent gelatin-coated coverslips
    The objective of this step is to perform a gelatin coating as homogeneous as possible. To achieve that, it is better to coat no more than 4 coverslips at a time, repeating the process as many times as necessary to obtain the desired final number of coverslips. By doing that, the fluorescent gelatin stock solution is used recently warmed for each group of four coverslips. Also, it is important to work reasonably quickly (through steps A1 to A4 in Figure 1) to avoid that the gelatin starts drying before it has formed a thin uniform coating of the coverslip.
    1. Sterilize coverslips by soaking them in 70% Ethanol solution, let them air dry. Gelatin-coated coverslips can be prepared in batches of 12 or 24 (one or two multiwell plates) and stored in the dark at 4 °C for up to 1-2 weeks.
    2. Prepare fluorescent gelatin solution by diluting the gelatin stock (prepared according to manufacturer’s instructions at 1 mg/ml by adding 5 ml of distilled water to the commercial vial), in PBS containing 2% sucrose. Typically, gelatin is used at a final concentration of 0.2 mg/ml. Warm the fluorescent gelatin stock at 60 °C prior to dilution and keep the working solution protected from light and warmed during the coating process. Prepare around 500 μl of fluorescent gelatin working solution for 24-coverslip batch. Diluted fluorescent gelatin solution can be reused once upon frozen storage.
    3. Cut a piece of parafilm of around 4 x 2 inches and tape it to the bench top. This will facilitate quick manipulation of the coverslips. Place a set of 4 coverslips in line on top of the parafilm. Have the gelatin working solution pre-warmed in-hand (i.e. by having a thermomixer or a water bath by your side on the bench top). Pipet 100 μl of warmed fluorescent gelatin solution on top of the first coverslip and return the stock to 60 °C (step A1 in Figure 1). Using forceps, lift the coverslip with the gelatin on top and, keeping it horizontal, use the other hand to spread the gelatin with the outside of the same pipet tip attached to a pipettor (step A2 in Figure 1). When spread, hold this coverslip over the next one to prepare and tilt the coverslip to a vertical position letting the excess of gelatin to fall on the next coverslip (step A3 in Figure 1). Still holding the first coverslip in a vertical position, use a soft vacuum source (only turned on lightly, just enough to hold a pipet tip at the end of the hose) to aspirate the extra-coating left on the bottom edge of the coverslip (step 4 in Figure 1). Using stronger vacuum (completely turned on) is not advised since it will leave a gelatin layer that is too thin. Place the coverslip inside the well of a 12-well plate protected from light. Immediately proceed to repeat the same procedure with the next coverslip.


      Figure 1. Steps for coating coverslips with fluorescent gelatin. 1-Add pre-warmed gelatin solution to the tip of sterilized coverslips; 2-Hold coverslip with forceps and spread gelatin with the help of a pipet tip. 3-Remove the excess gelatin solution over the next coverslip to prepare. 4-Carefully aspirate the excess gelatin from the lower side of the coverslip. Refer to text for further details.

    4. When all coverslips are coated, let them dry (coating will look whitish when dry).
    5. Add 1 ml of a pre-chilled solution of glutaraldehyde (diluted from the stock solution at 0.5% in PBS) to each well and incubate for 15 min on ice.
    6. Remove the glutaraldehyde and dispose properly. Wash coverslips three times at room temperature with PBS.
    7. Add 1 ml of a freshly prepared solution of Sodium Borohydride (5 mg/ml in PBS) and incubate for 3 minutes at room temperature. Stir the plate if necessary to avoid that the Hydrogen bubbles lift the coverslips to the surface of the liquid.
    8. Remove the Sodium Borohydride and dispose properly. Wash coverslips three times at room temperature with PBS.
    9. Transfer plates to a tissue culture hood. Using the forceps (an old pair of forceps with a bent tip may help in this process), transfer coverslips to a sterile 12-well plate and wash three times with sterile PBS. Coverslips may be used the same day or stored in PBS containing 200 units per ml of penicillin and 200 μg per ml of streptomycin (1:50 from the stock solution) at 4 °C in the dark for up to 2 weeks.
      Notes:
      1. An alternative method to cover the glass coverslips with the gelatin solution consists on pipetting the gelatin over the parafilm and invert the glass coverslip over the gelatin. This method may not achieve the same homogeneity but it may be easier for manipulation of coverslips.
      2. Using up to 1 mg/ml of fluorescently labeled gelatin solution to coat glass coverslips might help to increase the number of invadopodia formed by certain cell lines. Some cells attach very strongly and “pull” the gelatin coating in addition to degrading it. The effect of pulling cannot be distinguished from the effect of degradation for quantification purposes on this assay. Using 1 mg/ml of fluorescently labeled gelatin solution to coat glass coverslips or decreasing FBS in the growing medium might decrease the ability of some cells to “pull” the gelatin coating.

  2. Performing the fluorescent gelatin degradation assay
    1. Working under sterile conditions, transfer the desired number of coated coverslips to a new sterile 12-well plate. If the fluorescent gelatin coated coverslips were already stored in antibiotic-containing PBS, wash them three times with sterile PBS and incubate in 1 ml of the same complete growth medium that you will use in the experiment. Place them inside the 37 °C tissue culture incubator.
    2. Collect the cells to use in the assay using standard techniques (i.e. trypsinization), and count them using the hemocytometer or other suitable method.
    3. Plate 1 ml of a cell suspension, typically containing between 20-40,000 cells on top of each well containing 1 ml of medium.
    4. Return cells to the incubator for 8-16 h. The length of the assay should be determined empirically for each cell line and conditions.

  3. Processing cells for invadopodia detection and fluorescent microscopy
    1. At endpoint, wash cells once with PBS and quickly fix in a formaldehyde solution (4% in PBS) for 10-15 minutes at room temperature and protected from light.
    2. Remove formaldehyde and dispose properly. Wash three times with PBS and incubate in a BSA solution (3% in PBS containing 0.1% Triton X-100) for 15-30 min at room temperature and protected from light.
    3. Remove BSA solution and stain F-actin with Alexa Fluor 568 Phalloidin (diluted around 1:500 to 1:100 in PBS containing 0.3% BSA and 0.1% Triton X-100). Incubate for 0.5 to 1 h at room temperature protected from light. The optimal phalloidin dilution and incubation time depends on the cell line used and should be determined empirically.
    4. Remove the phalloidin solution and wash three times with PBS containing 0.1% Triton-X100 and three times with PBS alone.
    5. Mount the coverslips by inverting them over a glass slide containing a drop of mounting medium containing DAPI. Slides can be stored in the dark at 4 °C for several weeks.

  4. Imaging and quantifying invadopodia formation and activity
    1. Slides can be imaged with a fluorescent microscope equipped for detection of Alexa 488, Alexa 568 and DAPI. Invadopodia are detected in the “red” channel as F-actin rich puncta in the ventral surface of the cell in contact with the gelatin (Figure 2 C). Gelatin degradation is detected in the “green” channel as dark areas over the green background (Figure 2 B). Typically, a subset of F-actin rich structures will co-localize with discrete gelatin degradation spots, indicating that these F-actin rich structures are likely invadopodia (Figure 2 A).
    2. Quantification of invadopodia formation and activity may be performed on the same set of digital images. To perform a representative image collection, it is advisable to image areas that are representative to the whole coverslip in order to account for differences in the thickness of the gelatin coating. A possibility is to divide the coverlip into three imaginary columns and image five rows per column covering the whole height of each column. At least 15 fields per coverslip should be imaged, typically under 40x magnification taking all three channels (“red”, “green” and “blue”). Moving from one field to the next when in the channel for DAPI will help you identify areas containing similar number of cells while you are “blinded” for any other experimental outcome in a particular field to be imaged.


      Figure 2. Representative images of invadopodia and gelatin degradation assay. Digital images from a gelatin degradation assay performed on SCC61 cells. Images were obtained using a 40x objective in a fluorescence microscope. A. Merged channels showing fluorescent gelatin (green), F-actin staining (red) and nuclei (blue). B. Green channel (gelatin). C. Red channel (F-actin). D. Blue channel (nuclei). Arrowheads point to invadopodia in A and C.

    3. To quantify invadopodia formation, count the number of cells forming invadopodia on each digital image and normalize to the number of total cells in that same image. After counting all your images, collected data may be represented as “percent of cells forming invadopodia”. Perform the appropriate statistical analysis to determine significant differences among treatments.
    4. To quantify invadopodia activity, black and white images of gelatin degradation are analyzed using ImageJ (NIH). The objective is to measure “area fraction” (the percent of area that corresponds to degradation) on a given image. The “area fraction” value will then be normalized to the number of nuclei in each image as measured from the DAPI channel in the same field (Figure 3). The steps for obtaining the “area fraction” value are:
      1. Set Image J to measure “area fraction”: Analyze>Set Measurements>select “Area Fraction”.
      2. Open a black and white image of gelatin degradation (green channel) in Image J and then go to Image>Adjust>Threshold. If the black areas over white background are representative of the degradation in the original image, no further adjustment is necessary. If the program detects black areas that are broader than the actual degradation (i.e. because of irregular gelatin coating), the threshold has to be manually adjusted. On the new window move the bottom on the lower bar towards the left until the dark areas are representative of the real degradation observed in the original image.
      3. When the final image is representative of the gelatin degradation on the original image (as in Figure 3 C) click “set”. Measure “area fraction” by: Analyze>Measure. The values obtained in Image J may be exported to Microsoft Excel.
        The measurements corresponding “to area fraction” are normalized to the number of nuclei in the DAPI channel image from the same field. The final value corresponds to “normalized degradation”. The same analysis is repeated for each image of each sample. Values can be represented as mean and standard deviations. Perform the appropriate statistical analysis to determine significant differences among treatments. An example is showed in Figure 3.
        Note: When assessing the formation of invadopodia in your cell line of interest for the first time, it is advisable to perform additional immunofluorescent experiments to assess the presence of invadopodia components such as Tks5, cortactin and Arp2/3 at the F-actin rich structures.


        Figure 3. Quantification of invadopodia activity. Images from the green channel (gelatin) and blue channel (nuclei) from the same microscopy field are used. The original image from the green channel (A) is processed using Image J to obtain the image in (B), and the area fraction value. The original image from the blue channel (C) is used to calculate cell number. These two values are used to obtain the “normalized degradation” value of each microscopy field from a sample.

Acknowledgments

This protocol was initially adapted from: Mueller et al. (1992). The first adaptation was published in: Berdeaux et al. (2004). It was later implemented in: Díaz et al. (2013). Funding during first adaptation: National Institutes of Health grant CA17542 to G.S. Martin. Funding during implementation: National Institutes of Health CA129686 to S. A. Courtneidge. Other acknowledgements: The author would like to acknowledge Dr. G.S. Martin (UC Berkeley, California) and S.A. Courtneidge (UHSU, Portland, Oregon) for their support over the time this protocol was adapted and implemented.

References

  1. Berdeaux, R. L., Diaz, B., Kim, L. and Martin, G. S. (2004). Active Rho is localized to podosomes induced by oncogenic Src and is required for their assembly and function. J Cell Biol 166(3): 317-323.
  2. Diaz, B., Yuen, A., Iizuka, S., Higashiyama, S. and Courtneidge, S. A. (2013). Notch increases the shedding of HB-EGF by ADAM12 to potentiate invadopodia formation in hypoxia. J Cell Biol 201(2): 279-292.
  3. Mueller, S. C., Yeh, Y. and Chen, W. T. (1992). Tyrosine phosphorylation of membrane proteins mediates cellular invasion by transformed cells. J Cell Biol 119(5): 1309-1325.

简介

该协议旨在量化侵袭过程的形成和活性。 Invadopodia是介导细胞附着和细胞外基质重塑的癌细胞阐述的突出结构。 这些结构有助于癌细胞入侵和转移的能力。 在该方案中,通过基于荧光显微镜的测定法同时评估和定量侵袭多巴的存在及其活性。

关键字:癌症, 入侵, 侵袭性伪足, 转移, 明胶

材料和试剂

  1. 细胞( 。 SCC61头颈癌细胞)
  2. 组织培养基( 。 DMEM)(Mediatech,目录号:10013CV)
  3. 无菌PBS
  4. 胰蛋白酶0.05%/EDTA(Life Technologies,目录号:25300054)
  5. 青霉素/链霉素溶液(Omega Scientific,目录号PS-20)
  6. 乙醇(Decon Labs,目录号:2801)
  7. Forceps Dumont#5(Fine Science Tools,目录号:11252-20)
  8. 25%戊二醛(Polysciences,目录号:00376-500)
  9. 蔗糖(Fisher Scientific,目录号:S3500)
  10. 硼氢化钠(Fisher Scientific,目录号:S67825)
  11. 16%甲醛(Electron Microscopy Sciences,目录号:15710)
  12. 来自猪皮肤,俄勒冈绿488缀合物(Life Technologies,目录号:G13186)的明胶
  13. Alexa Fluor 568鬼笔环肽(Life Technologies,目录号:A12380)
  14. 安装介质,带有DAPI的Vectashield(Vector Laboratories,目录号:H-1200)
  15. 牛血清白蛋白级分V(EMD Millipore,目录号:2980-1KG)
  16. Triton X-100(Promega corporation,目录号:H5141)
  17. Parafilm(Bemis公司,目录号:PM996)

设备

  1. 圆形18mm直径玻璃盖玻片(0.13-0.17mm厚度)(Carolina Biological Supply Company,目录号:633033)
  2. 显微镜载玻片(75×25mm)(Thermo Fisher Scientific,目录号:1255015)
  3. 12孔微孔板(Corning,目录号:3513)
  4. 热固定器或水浴设置在60°C
  5. 组织培养孵化器
  6. 血细胞计数器(LabSource,目录号:0267154)http://www.lifetechnologies.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/counting-cells- in-a-hemacytometer.html
  7. 荧光显微镜
  8. 真空源

程序

  1. 准备荧光明胶涂层盖玻片
    该步骤的目的是进行尽可能均匀的明胶涂层。为了实现这一点,最好一次涂覆不超过4个盖玻片,重复该过程根据需要多次以获得所需的最终数量的盖玻片。通过这样做,荧光明胶储备溶液最近被用于每组四个盖玻片。此外,重要的是合理快速地工作(通过图1中的步骤A1至A4),以避免明胶在形成薄的均匀的盖玻片之前开始干燥。
    1. 通过将盖玻片浸泡在70%乙醇溶液中消毒盖玻片,让它们空气干燥。明胶包被的盖玻片可以分批制备12或24(一个或两个多孔板),并在黑暗中在4℃下储存长达1-2周。
    2. 通过在含有2%蔗糖的PBS中稀释明胶原料(根据制造商的说明书,1mg/ml,通过向商业小瓶中加入5ml蒸馏水制备)制备荧光明胶溶液。通常,明胶以0.2mg/ml的终浓度使用。在稀释前将荧光明胶原料温度升至60°C,并保持工作溶液免受光照,并在涂布过程中保温。准备大约500微升荧光明胶工作溶液为24盖玻片批次。稀释的荧光明胶溶液在冷冻储存后可重复使用一次。
    3. 切一块约4 x 2英寸的石蜡膜,并将其贴在台面上。这将便于快速操作盖玻片。放置一套4盖玻片在线在顶部的parafilm。让明胶工作溶液预先温热( 通过在台面上有一个恒温混合器或水浴)。吸取100微升温暖的荧光明胶溶液在第一个盖玻片的顶部,并将股票返回到60°C(图1中的步骤A1)。使用镊子,提起盖玻片与明胶在顶部,并保持水平,使用另一只手将明胶与移液器的同一个移液器吸头的外部(图1中的步骤A2)。当展开时,将该盖玻片保持在下一盖玻片上以准备并将盖玻片倾斜至垂直位置,使过量的明胶落在下一盖玻片上(图1中的步骤A3)。仍然在垂直位置握住第一个盖玻片,使用一个软真空源(只打开轻轻,刚好足以保持在软管末端的移液器吸头)吸出盖玻片底部边缘的额外涂层图1中的步骤4)。不建议使用更强的真空(完全打开),因为它会留下太薄的明胶层。将盖玻片放在一个12孔板的光保护的井内。立即进行重复相同的程序与下一个盖玻片。


      图1.用荧光明胶涂覆盖玻片的步骤。 1 - 将预热的明胶溶液加入灭菌盖玻片的尖端; 2 - 用钳子盖住盖玻片,并用移液管吸头扩散明胶。 3 - 除去过量的明胶溶液在下一个盖玻片上以准备。 4 - 小心地从盖玻片的下侧吸出过量的明胶。有关详细信息,请参阅文本。

    4. 当所有盖玻片涂层时,让它们干燥(干燥时涂层看起来发白)
    5. 向每个孔中加入1ml预冷的戊二醛溶液(从PBS中的0.5%储备溶液稀释),并在冰上孵育15分钟。
    6. 取出戊二醛并正确处理。在室温下用PBS洗涤盖玻片三次。
    7. 加入1ml新鲜制备的硼氢化钠溶液(5mg/ml的PBS溶液),并在室温下孵育3分钟。如果需要,搅拌板以避免氢气泡将盖玻片提升到液体表面
    8. 除去硼氢化钠并正确处理。在室温下用PBS洗涤盖玻片三次。
    9. 转移板组织培养罩。使用镊子(具有弯曲的尖端的老一对镊子可以在这个过程中可能有帮助),将盖玻片转移到无菌的12孔板,用无菌PBS洗涤三次。盖玻片可以在同一天使用,或者在4℃下在黑暗中储存在含有200单位/ml青霉素和200μg/ml链霉素(1:50的储备溶液)的PBS中达2周。 > 注意:
      1. 用明胶溶液覆盖玻璃盖玻片的替代方法在于将明胶吸移在石蜡膜上并将玻璃盖玻片翻转在明胶上。这种方法可能无法达到相同的均匀性,但它可能更容易操作盖玻片。
      2. 使用高达1mg/ml的荧光标记的明胶溶液涂覆玻璃盖玻片可能有助于增加某些细胞系形成的invadopodia的数目。除了降解明胶外,一些细胞非常强烈地附着并"拉"明胶涂层。牵引的效果不能与用于定量目的的降解效应区别于该测定。使用1mg/ml荧光标记的明胶溶液在生长培养基中涂覆玻璃盖玻片或降低FBS可能降低一些细胞"拉"明胶涂层的能力。

  2. 进行荧光明胶降解测定
    1. 在无菌条件下工作,将所需数量的涂层盖玻片转移到新的无菌12孔板中。如果荧光明胶包被的盖玻片已经存储在含抗生素的PBS中,用无菌PBS洗涤三次,并孵育在1ml相同的完整生长培养基,您将在实验中使用。将它们放在37℃组织培养箱中
    2. 收集细胞用于使用标准技术(即胰蛋白酶消化)的测定,并使用血细胞计数器或其他合适的方法计数。
    3. 板1ml的细胞悬浮液,通常含有20-40,000个细胞在每个孔的顶部含有1ml培养基。
    4. 将细胞返回培养箱中8-16小时。对于每种细胞系和条件,应该根据经验确定测定的长度
  3. 处理细胞的invadopodia检测和荧光显微镜
    1. 在终点,用PBS洗涤细胞一次,并在室温下快速固定在甲醛溶液(4%在PBS中)10-15分钟,并避光。
    2. 除去甲醛,正确处理。用PBS洗涤三次,并在BSA溶液(3%在含有0.1%Triton X-100的PBS中)在室温下孵育15-30分钟并避光。
    3. 去除BSA溶液和用Alexa Fluor 568鬼笔环肽(在含有0.3%BSA和0.1%Triton X-100的PBS中以约1:500至1:100稀释)染色F-肌动蛋白。在室温下孵育0.5至1小时,避光。最佳的鬼笔环肽稀释和孵育时间取决于所使用的细胞系,并且应该根据经验确定
    4. 除去鬼笔环肽溶液,用含有0.1%Triton-X100的PBS洗涤三次,用PBS单独洗涤三次。
    5. 通过翻转盖玻片在含有一滴含有DAPI的封固剂的载玻片上来安装盖玻片。幻灯片可以在4°C的黑暗中储存数周。

  4. 成像和量化invadopodia形成和活动
    1. 载玻片可以用配备用于检测Alexa 488,Alexa 568和DAPI的荧光显微镜成像。在"红色"通道中,在与明胶接触的细胞的腹侧表面中检测到不连续胚胎为富含肌动蛋白的点(图2C)。在"绿色"通道中检测到明胶降解作为绿色背景上的黑色区域(图2B)。通常,富含F-肌动蛋白的结构的子集将与离散明胶降解点共定位,表明这些富含F-肌动蛋白的结构可能是invadopodia(图2A)。
    2. 可以在同一组数字图像上进行对隐匿形成和活性的定量。为了执行代表性图像收集,建议对代表整个盖玻片的区域进行成像,以便解决明胶涂层的厚度差异。一种可能性是将盖玻片分成三个假想列,并且每列覆盖每列的整个高度五行。每个盖玻片至少15个视野应该被成像,通常在40倍放大,采取所有三个通道("红色","绿色"和"蓝色")。当在DAPI的通道中移动时,从一个字段移动到下一个字段将帮助您识别包含相似数量的单元格的区域,同时对被成像的特定区域中的任何其他实验结果"盲目化"。


      图2. invadopodia和明胶降解测定的代表性图像。在SCC61细胞上进行明胶降解测定的数字图像。在荧光显微镜中使用40x物镜获得图像。 A.合并的通道显示荧光明胶(绿色),F-肌动蛋白染色(红色)和细胞核(蓝色)。 B.绿色通道(明胶)。 C.红色通道(F-肌动蛋白)。蓝色通道(核)。箭头指向A和C中的invadopodia。

    3. 为了定量invadopodia形成,计数在每个数字图像上形成invadopodia的细胞数,并归一化到同一图像中的总细胞数。计数所有图像后,收集的数据可以表示为"形成invadopodia的细胞的百分比"。进行适当的统计分析以确定治疗之间的显着差异
    4. 为了定量invadopodia活动,使用ImageJ(NIH)分析明胶降解的黑白图像。目的是测量给定图像上的"面积分数"(对应于退化的面积百分比)。然后将"面积分数"值归一化为从相同字段中的DAPI通道测量的每个图像中的细胞核的数目(图3)。获得"面积分数"值的步骤是:
      1. 设置图像J以测量"面积分数":分析>设置测量>选择"面积分数"。
      2. 在图像J中打开明胶降解的黑白图像(绿色通道),然后进入图像>调整>阈值。如果白色背景上的黑色区域表示原始图像中的劣化,则不需要进一步调整。如果程序检测到比实际劣化更宽的黑色区域(即,由于不规则的明胶涂层),则必须手动调节阈值。在新窗口上,将底部条上的底部向左移动,直到暗区表示在原始图像中观察到的真实退化。
      3. 当最终图像代表原始图像上的明胶降解时(如图3C所示),点击"设置"。通过以下方法测量"面积分数":分析>测量。在图像J中获得的值可以导出到Microsoft Excel。
        对应于"面积分数"的测量被归一化为来自相同字段的DAPI通道图像中的核的数量。最终值对应于"归一化退化"。对每个样品的每个图像重复相同的分析。值可以表示为平均值和标准偏差。进行适当的统计分析,以确定治疗之间的显着差异。示例如图3所示。
        注意:当首次评估感兴趣的细胞系中invadopodia的形成时,建议进行额外的免疫荧光实验以评估invadopodia组分如Tks5,cortactin和Arp2/3在F-肌动蛋白丰富的结构。


        图3.使用来自相同显微镜视野的来自绿色通道(明胶)和蓝色通道(核)的图像。使用图像J处理来自绿色通道(A)的原始图像,以获得(B)中的图像和面积分数值。来自蓝色通道(C)的原始图像用于计算细胞数目。这两个值用于从样品获得每个显微镜视野的"归一化退化"值

致谢

该方案最初改编自Mueller等人(1992)。第一个改编发表于:Berdeaux等人(2004)。它后来在Díaz等人(2013)中实现。 第一次适应期间的资金:国立卫生研究院授予CA17542到G.S. Martin。实施过程中的资金:国家卫生研究院CA129686至S.A. Courtneidge。其他致谢:作者要感谢G.S. Martin(加利福尼亚州伯克利分校)和S.A. Courtneidge(UHSU,Portland,Oregon)在本协议修改和实施期间给予的支持。

参考文献

  1. Berdeaux,R.L.,Diaz,B.,Kim,L。和Martin,G.S。(2004)。 活性Rho定位于由致癌Src诱导的podosomes,并且是其组装和功能所需的。 a> J Cell Biol 166(3):317-323
  2. Diaz,B.,Yuen,A.,Iizuka,S.,Higashiyama,S.and Courtneidge,S.A。(2013)。 Notch增加了ADAM12对HB-EGF的脱落,以增强缺氧中的invadopodia形成。 J Cell Biol 201(2):279-292
  3. Mueller,S.C.,Yeh,Y。和Chen,W.T。(1992)。 膜蛋白的酪氨酸磷酸化介导转化细胞的细胞侵袭。细胞 Biol 119(5):1309-1325
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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: © 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. Díaz, B. (2013). Invadopodia Detection and Gelatin Degradation Assay. Bio-protocol 3(24): e997. DOI: 10.21769/BioProtoc.997.
  2. Diaz, B., Yuen, A., Iizuka, S., Higashiyama, S. and Courtneidge, S. A. (2013). Notch increases the shedding of HB-EGF by ADAM12 to potentiate invadopodia formation in hypoxia. J Cell Biol 201(2): 279-292.
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