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[Bio101] Immunofluorescence Detection /F-actin Staining of MTLn3 Cells
[Bio101] MTLn3细胞的免疫荧光检测/F-actin染色   

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Abstract

Epidermal growth factor (EGF)-stimulated MTLn3 cells protrusion play an important role in cell migration. Phalloidin which binds F-actin in cells is an imaging tool used with light microscopy to investigate the distribution of actin. The protocol described here can be useful for observing signaling in the EGF pathway.

Materials and Reagents

  1. MTLn3 cells
  2. Type I rat tail collagen (BD Biosciences, catalog number: 354236 )
  3. Leibovitz's L15 media w/o phenol red (Life Technologies, InvitrogenTM, catalog number: 21083-027 )
  4. Leibovitz's L15 media w/ phenol red (Life Technologies, InvitrogenTM, catalog number: 11415-064 )
  5. Rhodamin phalloidin red Cy3 (Life Technologies, InvitrogenTM, catalog number: R415 )
  6. Phosphate buffered saline (PBS)
  7. Trypsin-EDTA
  8. HCI
  9. 95% ethanol
  10. Acetic acid
  11. FBS-alpha-MEM
  12. Paraformaldehyde
  13. Triton X-100
  14. Glycine
  15. BSA
  16. N-propyl gallate
  17. Glycerol
  18. Methanol
  19. Nail polish
  20. Collagen solution
  21. EGF (EMD Millipore, catalog number: 01-102 ) (see Recipes)
  22. Rhodamine phalloidin (see Recipes)

Equipment

  1. Centrifuges
  2. Coverslips
  3. Tissue culture flask
  4. Parafilm
  5. Culture dish
  6. Humidified chamber
  7. Sharp forceps
  8. Water bath
  9. Culture hood

Procedure

  1. Collagen coated coverslip preparation:
    1. Wash coverslips twice with 95% ethanol.
    2. Wash and incubate the coverslips in IN HCI for 1 h at room temperature (RT).
    3. Wash twice with sterilized PBS (coverslips can be stored in methanol in bulk).
    4. If taken straight from methanol, wash twice with sterilized PBS to rehydrate the coverslips. From this point on every procedure needs to be conducted under sterile conditions.
    5. Monolayer the coverslips on 100 mm culture dish.
    6. Coat the coverslips in acetic acid-collagen solution:
      256 μl of 3.33 m ml-1 type I collagen into 20 ml of filter sterilize 0.01 M acetic acid. Add 20 ml of acetic acid collagen solution to the monolayer coverslips in 100 mm dish. Incubate in the culture hood at RT for 2 h.
    7. After collagen coating, rinse once gently with sterilized PBS. At this point the coverslips can be stored in PBS at 4 °C for 1 week.

  2. Splitting MTLn3 cells on collagen coated coverslips and starvation:
    1. For 80 mm tissue culture flask, MTLn3 cells should be kept 50-70% confluent at all times. Over confluent cells do not respond to EGF stimulation well.
    2. Rinse once with 1 ml trypsin-EDTA, aspirate and then trypsinize with 1 ml trypsin-EDTA at 37 °C incubator for 3-5 min until cells come off the flask.
    3. Add 9 ml 5% FBS-alpha-MEM into the flask. Pipet the cells up and down a few times to break the cell clumps.
    4. From a 70% confluent 80 mm flask, take 3-5 ml cells to one 100 mm culture dish of the collagen coated coverslips. Usually it takes > 24 h for MTLn3 cells to be well spread on the coverslips.
    5. Wash once with warm starvation media, 0.35% BSA-L15 media, and starve the cells in this media for 3 h at 37 °C without CO2.

  3. EGF stimulation and cell fixation:
    1. Place culture plate into 37 °C water bath. Add appropriate volume of EGF stimulation buffer to the plate and stimulated for 3 min.
    2. Fix cell with warm 4% Paraformaldehyde in 37 °C water bath for 10 min.
    3. After fixation, rinse 1x with PBS and the coverslips can be stored in PBS at 4 °C overnight.

  4. Immunofluorescence/ F-actin staining
    1. Permeablize the fixed cells with 0.5% triton X-100-PBS for 10 min at RT.
    2. Optional: rinse once with 0.1 M glycine-PBS and incubate in 0.1 M glycine-PBS for 10 min at RT to reduce background.
    3. Block with 1% FBS- 1% BSA- PBS at RT 4x 5 min, then transfer coverslips onto parafilm.
    4. Incubate 1-2 h with primary antibody (50 μl per coverslips) usually start with 1:200 dilution in 1% BSA-PBS buffer in a humidified chamber.
    5. Wash with PBS or 1% BSA-PBS 5 min 3-5 times to wash out non-specific primary antibody.
    6. Incubate with 1: 200 secondary antibody in 1% BSA-PBS for 1 h at RT.
    7. Aspirate the secondary antibody, and incubate 20 min with Rhodamine phalloidin (1:15 dilution in 1% BSA-PBS)
    8. Wash the coverslips with PBS 5 times for 5 min each.

  5. Mounting coverslips:
    1. Making mounting buffer (0.11 g n-propyl gallate + 2.5 ml 2x PBS + 2.5 ml glycerol), vortex until all of it has dissolved (usually takes 40 min). Store in dark for 2 weeks.
    2. Add 3 μl mounting solution on the slide. Use sharp forceps to pick up the coverslip, absorb excess buffer on the edge of the coverslips, carefully drop the coverslip on mounting solution.
    3. Apply nail polish on four corners of the coverslip and seal the edge with nail polish all round. Store the slides either in -20 °C or 4 °C in slide boxes.

Recipes

  1. To make EGF aliquots
    Spin down the EGF powder, add 333 μl PBS and then aliquot into either 5 μl or 10 μl aliquots. Store in -80 °C.
  2. To make Rhodamine phalloidin stock
    Spin down the power and add 1.5 ml methonol to dissolve. Store in -20 °C in the dark.

References

  1. Capani, F., Deerinck, T. J., Ellisman, M. H., Bushong, E., Bobik, M. and Martone, M. E. (2001). Phalloidin-eosin followed by photo-oxidation: a novel method for localizing F-actin at the light and electron microscopic levels. J Histochem Cytochem 49(11): 1351-1361.
  2. Yip, S. C., El-Sibai, M., Coniglio, S. J., Mouneimne, G., Eddy, R. J., Drees, B. E., Neilsen, P. O., Goswami, S., Symons, M., Condeelis, J. S. and Backer, J. M. (2007). The distinct roles of Ras and Rac in PI 3-kinase-dependent protrusion during EGF-stimulated cell migration. J Cell Sci 120(Pt 17): 3138-3146.

简介

表皮生长因子(EGF)刺激的MTLn3细胞突起在细胞迁移中发挥重要作用。 在细胞中结合F-肌动蛋白的鬼笔环肽是用于光学显微镜以研究肌动蛋白分布的成像工具。 本文所述的方案可用于观察EGF途径中的信号传导。

材料和试剂

  1. MTLn3细胞
  2. I型大鼠尾胶原(BD Biosciences,目录号:354236)
  3. Leibovitz's L15培养基w/o酚红(Life Technologies,Invitrogen TM ,目录号:21083-027)
  4. Leibovitz's L15培养基w /酚红(Life Technologies,Invitrogen TM ,目录号:11415-064)
  5. 罗丹明鬼笔环肽红Cy3(Life Technologies,Invitrogen TM,目录号:R415)
  6. 磷酸盐缓冲盐水(PBS)
  7. 胰蛋白酶-EDTA
  8. HCI
  9. 95%乙醇
  10. 乙酸
  11. FBS-α-MEM
  12. 多聚甲醛
  13. Triton X-100
  14. 甘氨酸
  15. BSA
  16. 没食子酸丙酯
  17. 甘油
  18. 甲醇
  19. 指甲油
  20. 胶原蛋白溶液
  21. EGF(EMD Millipore,目录号:01-102)(参见Recipes)
  22. 罗丹明鬼笔环肽(参见配方)

设备

  1. 离心机
  2. 盖舌
  3. 组织培养瓶
  4. parafilm
  5. 文化菜
  6. 加湿室
  7. 锋利的镊子
  8. 水浴
  9. 文化宫

程序

  1. 胶原覆盖的盖玻片制剂:
    1. 用95%乙醇洗涤盖玻片两次。
    2. 洗涤并孵育盖玻片在1N HCl在室温(RT)1小时。
    3. 用无菌PBS洗涤两次(盖玻片可以大量储存在甲醇中)
    4. 如果直接从甲醇中取出,用无菌PBS洗涤两次 再水化盖玻片。 从这一点上来说,每个过程都需要   在无菌条件下进行
    5. 单层盖在100毫米培养皿盖玻片
    6. 在乙酸 - 胶原溶液中涂布盖玻片:
      将256μl3.33ml ml -1 I型胶原加入20ml过滤灭菌中 0.01M乙酸。 加入20ml乙酸胶原溶液 单层盖玻片在100mm皿中。 在室温下在培养罩中孵育 2小时。
    7. 胶原蛋白涂层后,轻轻冲洗一次 无菌PBS。 在这一点上,盖玻片可以在4℃储存在PBS中 ℃1周。

  2. 分裂MTLn3细胞在胶原涂层的盖玻片和饥饿:
    1. 对于80mm组织培养瓶,MTLn3细胞应保持50-70% 汇合在任何时候。 在汇合的细胞上不响应EGF 刺激井
    2. 用1ml胰蛋白酶-EDTA冲洗一次,吸出和 然后用1ml胰蛋白酶-EDTA在37℃培养箱中胰蛋白酶消化3-5分钟 直到细胞从瓶中流出
    3. 加入9 ml 5%FBS-α-MEM到烧瓶中。 吸取细胞上下数次,打破细胞团块。
    4. 从70%汇合的80mm烧瓶中,取3-5ml细胞至一个100mm 培养皿的胶原包被的盖玻片。 通常需要> 24   h,MTLn3细胞在盖玻片上良好扩散。
    5. 用温热的饥饿培养基(0.35%BSA-L15培养基)洗涤一次,并在37℃,不含CO 2的条件下使细胞在该培养基中饥饿3小时。

  3. EGF刺激和细胞固定:
    1. 将培养板置于37℃水浴中。 加入适当体积的EGF   刺激缓冲液到板上并刺激3分钟
    2. 用温热的4%多聚甲醛在37℃水浴中固定细胞10分钟。
    3. 固定后,用PBS冲洗1次,盖玻片可以储存在4℃的PBS过夜。

  4. 免疫荧光/F-肌动蛋白染色
    1. 在室温下用0.5%triton X-100-PBS渗透固定的细胞10分钟。
    2. 可选:用0.1 M甘氨酸-PBS冲洗一次,并在0.1 M甘氨酸-PBS中孵育10分钟,以减少背景。
    3. 用1%FBS-1%BSA-PBS在室温下封闭4x 5分钟,然后将盖玻片转移到石蜡膜上。
    4. 孵育1-2 h与一级抗体(50μl每盖玻片)通常   开始于在加湿室中在1%BSA-PBS缓冲液中的1:200稀释。
    5. 用PBS或1%BSA-PBS洗涤5次,每次3-5次,洗去非特异性一抗
    6. 与1:200二抗在1%BSA-PBS中孵育1小时。
    7. 吸出第二抗体,与罗丹明鬼笔环肽(1:15稀释在1%BSA-PBS)孵育20分钟
    8. 用PBS洗涤盖玻片5次,每次5分钟。

  5. 安装盖玻片:
    1. 制备安装缓冲液(0.11g没食子酸正丙酯+ 2.5ml 2×PBS +   甘油),涡旋直至其全部溶解(通常需要40分钟)。   在黑暗中储存2周。
    2. 加入3μl安装溶液 滑动。 使用锋利的镊子拿起盖玻片,吸收多余的缓冲区 在盖玻片的边缘,小心地将盖玻片上安装 解。
    3. 应用指甲油在盖玻片的四个角落 并用指甲油全面密封边缘。 将幻灯片存储在   -20℃或4℃。

食谱

  1. 使EGF等分试样
    向下旋转EGF粉末,加入333μlPBS,然后等分到5μl或10μl等分试样。 储存于-80°C。
  2. 制备罗丹明鬼笔环肽原料
    旋下粉末,加入1.5 ml甲醇溶解。 在-20°C避光储存。

参考文献

  1. Capani,F.,Deerinck,T.J.,Ellisman,M.H.,Bushong,E.,Bobik,M.and Martone,M.E。(2001)。 鬼笔环肽曙红其次是光氧化:一种新颖的方法,用于定位F-肌动蛋白在光和 电子显微镜水平。 J Histochem Cytochem 49(11):1351-1361。
  2. Yip,SC,El-Sibai,M.,Coniglio,SJ,Mouneimne,G.,Eddy,RJ,Drees,BE,Neilsen,PO,Goswami,S.,Symons,M.,Condeelis,JS和Backer,JM 2007)。 在EGF刺激的细胞迁移过程中Ras和Rac在PI 3-激酶依赖性突出中的不同作用 。 J Cell Sci 120(Pt 17):3138-3146。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Pang, H. (2012). Immunofluorescence Detection /F-actin Staining of MTLn3 Cells. Bio-protocol Bio101: e206. DOI: 10.21769/BioProtoc.206;
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