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Extravillous Trophoblast Migration and Invasion Assay
绒毛外滋养层细胞的迁移和侵袭试验   

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Abstract

Extravillous trophoblast (EVT) migration and invasion through the decidualized endometrium is essential to successful placentation. SGHPL-4 cells, an EVT cell line derived from first trimester placenta, is a widely used model of cytotrophoblast differentiation into an invasive phenotype. Here we describe a quantitative cell migration assay that can be modified to also measure cell invasion. SGHPL-4 cells were seeded into BD Fluoroblok cell culture inserts constructed with an 8 μm porous membrane and allowed to migrate towards epidermal growth factor, a known chemoattractant for EVTs. To assess EVT invasion, Fluoroblok inserts were first coated with Matrigel, a basement membrane matrix. SGHPL-4 cells were labeled with calcein AM and cells that had invaded and/or migrated across the membrane were quantified by a bottom-reading fluorescence plate reader. The advantage of the Fluoroblok inserts over other migration/invasion assays is that they allow nondestructive detection of migrated cells.

Materials and Reagents

  1. SGHPL-4 cells (Kindly provided by Dr. Guy Whitley, St. George’s University of London)
  2. Ham’s F10 Nutrient Mix (Life Technologies, InvitrogenTM, catalog number: 11550-043 )
  3. Fetal bovine serum (FBS)
  4. Dulbecco’s Phosphate-Buffered Saline (DPBS) without Ca2+ and Mg2+ (Life Technologies, InvitrogenTM, catalog number: 14190 )
  5. TrypLE Express (Life Technologies, InvitrogenTM, catalog number: 12604013 )
  6. Matrigel, Growth Factor Reduced, Phenol Red Free (BD Biosciences, catalog number: 356231 )
  7. Recombinant Human Epidermal Growth Factor (hEGF) (BD Biosciences, catalog number: 354052 )
  8. BD Falcon HTS FluoroBlok Inserts (BD Biosciences, catalog number: 35112 )
  9. Calcein AM (Life Technologies, InvitrogenTM, catalog number: C3100MP )
  10. Hank’s balanced salt soution (HBSS) (Life Technologies, InvitrogenTM, catalog number: 14025 )

Equipment

  1. Centrifuge
  2. 37 °C, 5% CO2 Cell culture incubator
  3. Inverted Fluorescent Microscope
  4. Fluorescent plate reader

Procedure


    DAY 1

  1. For Invasion Assay, pre-Coat Fluoroblok Filter (8 μm porous membrane)
    1. Prechill Fluoroblok inserts, companion plates and pipet tips to help maintain Matrigel in the liquid state.
    2. Place desired number of prechilled inserts into a 24-well companion plate.
    3. Add 50 μl of 1:10 Matrigel (diluted in HamF10) to each transwell insert.
    4. Incubate at 37 °C, 3 h.
  2. Serum starve cultures (70-75% confluent) for 24 h in 0.5% FBS/HamF10
    1. Aspirate media.
    2. Wash with 7 ml warm DPBS (without Ca2+ and Mg2+).
    3. Add 12 ml warm 0.5% FBS/HamF10.
    4. Incubate cells for 24 h at 37 °C.


    DAY 2

  1. Prepare cells (Upper Chamber)
    1. Rinse cells once with 10 ml DPBS (without Ca2+ and Mg2+); add 3 ml TrypLE Express and incubate at 37 °C for 3-5 min; add 7 ml 0.5% FBS/HamF10 →10 ml total.
    2. Count cells using a hemacytometer.
    3. In a 50 ml conical tube, centrifuge cells at 300 x g for 10 min.
    4. Remove supernatant and resuspend cells in 0% FBS/HamF10 to obtain a cell suspension concentration of 1.2 x 106 cells/ml (or 1,250 cells/μl).
    5. Cap tube and store at room temperature till ready to load in chamber.
  2. Prepare the chemoattractant (Treatments in Bottom Chamber)
    1. Dilute desired chemoattractant in 0% FBS/HamF10. You will need 800 μl per well.
    2. Prepare 10 ng/ml EGF as positive control.
    3. Add 800 μl of chemoattractant to the bottom of each well. Avoid bubbles.
  3. Assemble invasion chamber
    1. Using a forceps, carefully remove insert from empty well.
    2. Add 200 μl of cells (2.5 x 105 for Invasion Assay or 5 x 104 for Migration Assay) to Matrigel-coated (for Invasion Assay) or uncoated insert (for Migration Assay).
    3. Lower the insert at an angle into the well containing the chemotactic substance. Check for bubbles by looking under the plate. If there are bubbles, remove insert and try again.
    4. Incubate at 37 °C for 12 h for Cell Migration Assay or 20-22 h for Cell Invasion Assay.


    DAY 3

  1. After invasion period, label invaded cells (on lower side of filter) with Calcein AM. For each well, add 2 μl of Calcein AM to 500 μl of HBSS.
  2. Carefully aspirate the media from the insert, without disturbing the Matrigel layer.
  3. Transfer the insert to a fresh well containing Calcein AM/HBSS solution.
  4. Incubate at 37 °C for 1 h in the dark.
  5. Read plate on fluorescent plate reader at 520 nm or take pictures using an epifluorescent microscope.

Acknowledgments

This protocol is adapted from Angelova et al. (2012).

References

  1. Angelova, M., Zwezdaryk, K., Ferris, M., Shan, B., Morris, C. A. and Sullivan, D. E. (2012). Human cytomegalovirus infection dysregulates the canonical Wnt/beta-catenin signaling pathway. PLoS Pathog 8(10): e1002959.
  2. LaMarca, H. L., Ott, C. M., Honer Zu Bentrup, K., Leblanc, C. L., Pierson, D. L., Nelson, A. B., Scandurro, A. B., Whitley, G. S., Nickerson, C. A. and Morris, C. A. (2005). Three-dimensional growth of extravillous cytotrophoblasts promotes differentiation and invasion. Placenta 26(10): 709-720. 
  3. Warner, J. A., Zwezdaryk, K. J., Day, B., Sullivan, D. E., Pridjian, G. and Morris, C. A. (2012). Human cytomegalovirus infection inhibits CXCL12- mediated migration and invasion of human extravillous cytotrophoblasts. Virol J 9: 255.

简介

Extravillous滋养层(EVT)迁移和通过蜕膜化子宫内膜的侵入是成功胎盘成形的关键。 SGHPL-4细胞,来自前三个月胎盘的EVT细胞系,是广泛使用的细胞滋养层分化为侵袭性表型的模型。在这里我们描述了一种定量细胞迁移实验,可以修改也测量细胞入侵。将SGHPL-4细胞接种到用8μm多孔膜构建的BD Fluoroblok细胞培养插入物中,并允许其向表皮生长因子(一种已知的EVTs化学吸引剂)迁移。为了评估EVT侵入,首先用Matrigel(基底膜基质)包被Fluoroblok插入物。用钙黄绿素AM标记SGHPL-4细胞,并通过底部读数荧光读板器定量已经侵入和/或迁移穿过膜的细胞。 Fluoroblok插入物优于其他迁移/侵袭测定的优点是它们允许迁移细胞的非破坏性检测。

材料和试剂

  1. SGHPL-4细胞(由Dr.Whiteley,St.George's University of London提供)
  2. Ham's F10营养混合物(Life Technologies,Invitrogen TM ,目录号:11550-043)
  3. 胎牛血清(FBS)
  4. 不含Ca 2+和Mg 2+的Dulbecco's磷酸盐缓冲盐水(DPBS)(Life Technologies,Invitrogen TM,目录号:14190) br />
  5. TrypLE Express(Life Technologies,Invitrogen TM ,目录号:12604013)
  6. Matrigel,Growth Factor Reduced,Phenol Red Free(BD Biosciences,目录号:356231)
  7. 重组人表皮生长因子(hEGF)(BD Biosciences,目录号:354052)
  8. BD Falcon HTS FluorBlok Inserts(BD Biosciences,目录号:35112)
  9. Calcein AM(Life Technologies,Invitrogen TM,目录号:C3100MP)
  10. Hank's平衡盐溶液(HBSS)(Life Technologies,Invitrogen TM ,目录号:14025)

设备

  1. 离心机
  2. 37℃,5%CO 2细胞培养箱
  3. 倒置荧光显微镜
  4. 荧光平板读取器

程序


    第1天

  1. 对于侵袭测定,预涂Fluoroblok过滤器(8μm多孔膜)
    1. Prechill Fluoroblok插入物,配套板和移液管吸头以帮助维持Matrigel处于液体状态。
    2. 将所需数量的预冷刀片放入24孔伴板中
    3. 添加50微升的1:10 Matrigel(稀释在HamF10)到每个transwell插入。
    4. 在37℃,3小时孵育
  2. 血清饥饿培养(70-75%汇合)在0.5%FBS/HamF10中24小时
    1. 吸出介质。
    2. 用7ml温热的DPBS(不含Ca 2+和Mg 2+ + )洗涤。
    3. 加入12ml温热的0.5%FBS/HamF10
    4. 在37℃下孵育细胞24小时


    第2天

  1. 准备细胞(上腔)
    1. 用10ml DPBS(不含Ca 2+和Mg 2+)冲洗细胞一次; 加入3ml TrypLE Express并在37℃孵育3-5分钟; 添加7ml 0.5%FBS/HamF10→ 10 ml 。
    2. 使用血细胞计数器计数细胞。
    3. 在50ml锥形管中,以300×g离心细胞10分钟
    4. 去掉   上清液并在0%FBS/HamF10中重悬细胞以获得细胞 悬浮液浓度为1.2×10 6个细胞/ml(或1250个细胞/μl)
    5. 盖管,并在室温下储存,直到准备装入室中
  2. 准备化学引诱物(底部处理)
    1. 在0%FBS/HamF10中稀释所需的化学引诱物。 您将需要每孔800μl。
    2. 准备10 ng/ml EGF作为阳性对照
    3. 向每个孔的底部添加800μl的化学引诱物。 避免气泡。
  3. 组装入侵室
    1. 使用镊子,小心地从空孔中取出插件。
    2. 将200μl细胞(用于侵袭测定的2.5×10 5个/对于迁移测定的5×10 4个)加入基质胶包被的(用于侵入测定)或未包被的插入物(用于 迁移测定)。
    3. 降低 该插入物以一定角度进入含有趋化性的孔中 物质。 通过在板下查看气泡。 如果有的话 气泡,请移除插入,然后重试。
    4. 在37℃孵育12小时用于细胞迁移测定或20-22小时用于细胞侵袭测定。


    第3天

  1. 在侵袭期后,标记物用钙黄绿素AM侵袭细胞(在滤膜的下侧)。 对于每个孔,将2μlCalcein AM加入500μlHBSS中
  2. 小心地从插入物中吸出介质,而不干扰基质胶层
  3. 将插入物转移到含有钙黄绿素AM/HBSS溶液的新鲜孔中
  4. 在37℃在黑暗中孵育1小时。
  5. 在荧光板读数器上在520nm读板或使用落射荧光显微镜拍照

致谢

该协议改编自Angelova等人(2012)。

参考文献

  1. Angelova,M.,Zwezdaryk,K.,Ferris,M.,Shan,B.,Morris,C.A。和Sullivan,D.E。 人巨细胞病毒感染会调节规范的Wnt /β-连环蛋白信号通路。 PLoS Pathog 8(10):e1002959。
  2. LaMarca,H.L.,Ott,C.M.,Honer Zu Bentrup,K.,Leblanc,C.L.,Pierson,D.L.,Nelson,A.B.,Scandurro,A.B.,Whitley,G.S.Nickerson,C.A.and Morris, 外膜细胞滋养层的三维生长促进分化和侵袭。胎盘 26(10):709-720。
  3. Warner,J.A.,Zwezdaryk,K.J.,Day,B.,Sullivan,D.E.,Pridjian,G.and Morris,C.A。(2012)。 人类巨细胞病毒感染抑制CXCL12介导的人类外膜细胞滋养层的迁移和侵袭。 Virol J 9:255。
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Copyright: © 2013 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. Angelova, M., Machado, H. L., Swan, K. F., Morris, C. and Sullivan, D. E. (2013). Extravillous Trophoblast Migration and Invasion Assay. Bio-protocol 3(15): e840. DOI: 10.21769/BioProtoc.840.
  2. Angelova, M., Zwezdaryk, K., Ferris, M., Shan, B., Morris, C. A. and Sullivan, D. E. (2012). Human cytomegalovirus infection dysregulates the canonical Wnt/beta-catenin signaling pathway. PLoS Pathog 8(10): e1002959.
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