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In vitro Tumor Cell Migration Assay Using ThinCertsTM (Transwells)
使用ThinCertsTM (一种侵袭迁移培养皿)进行体外肿瘤细胞迁移试验   

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参见作者原研究论文

本实验方案简略版
Clinical Cancer Research
Aug 2015

 

Abstract

The high migration rate of tumor cells often results in poor prognosis for the survival of the patients. Here, we describe a protocol to measure the migration of cells using a quantitative assay. The relative tumor cell migration was measured using ThinCertsTM cell culture inserts and a lactate dehydrogenase (LDH) assay to quantify the relative cell number. The quantification of the migration with the LDH kit is much more precise than other methods using i.e. crystal blue to count the cells.

Keywords: Migration assay (迁移实验), Transwells (Transwells), Tumor cells (肿瘤细胞), LDH assay (LDH法)

Materials and Reagents

  1. 12 well cell culture plate (Greiner Bio One International GmbH, catalog number: 665180 )
  2. 96 well tissue culture test plate (TPP Techno Plastic Products AG, catalog number: 92696 )
  3. 1.5 ml tubes (Carl Roth GmbH + Co., catalog number: 7080.1 )
  4. ThinCertsTM cell culture inserts with a pore diameter of 8 µm (Greiner Bio-One GmbH, catalog number: 665638 )
  5. Cover slips for haemocytometer (Carl Roth GmbH + Co., catalog number: L189.1 )
  6. Filter tips (10 µl, 20 µl, 100 µl, 200 µl and 1,000 µl) (Carl Roth GmbH + Co., catalog number: 771288 , 774288 , 772288 , 739288 and 740288 )
  7. Tumor cells (the lung cancer cell lines H1975 and 2106T)
  8. Cell culture medium (cell line specific), with and without fetal bovine serum (FBS)
  9. Heat-inactivated FBS (E.U.-approved, South America Origin) (Thermo Fischer Scientific, catalog number: 10500-064 )
  10. 1x Dulbecco’s phosphate-buffered saline (DPBS, no calcium, no magnesium) (Thermo Fischer Scientific, GibcoTM, catalog number: 14190-094 )
  11. StemPro® Accutase® cell dissociation reagent (Thermo Fischer Scientific, GibcoTM, catalog number: A11105-01 )
  12. Mitomycin C (AppliChem GmbH, catalog number: A2190,0002 )
  13. Trypan blue solution (Sigma-Aldrich, catalog number: T8154 )
  14. 10x cell lysis buffer (Cell Signaling Technology, catalog number: 9803S )
  15. Cytotoxicity detection kit (LDH) (Roche Diagnostics, catalog number: 11644793001 )

Equipment

  1. CO2 cell incubator (Panasonic Corporation, Sanyo, model: SANYO-InCu saFe® )
  2. Neubauer cell counting chamber (VWR International, catalog number: BRND717810 )
  3. Microcentrifuge (Eppendorf AG, model: 5417R )
  4. Vortex mixer (VWR International, catalog number: 444-1372 )
  5. Versatile microplate absorbance reader (Tecan Trading AG, model: sunriseTM )
  6. Gilson’s Pipetman classic pipettes ( P10 , P20 , P100 , P200 , P1000 )

Procedure


Figure 1. Flowchart of in vitro tumor cell migration assay

  1. Treat the cells with the desired assay (e.g., siRNA knockdown of a target gene or drug delivery) in 12 well cell culture plates.
  2. Replace the growth medium by 1 ml complete serum-free medium for 16 h overnight at 37 °C in the CO2 incubator.
  3. Treat the cells with 10 µg/ml mitomycin C in 1 ml serum-free medium for 2 h at 37 °C in the CO2 incubator to inhibit further cell proliferation.
  4. Wash cells once with 1 ml sterile 1x PBS.
  5. Add 250 µl accutase solution and incubate the cells in the incubator for approximately 5-10 min (depending on the adhesion of the used cells) to detach them.
  6. Add 1 ml serum-free medium to the cells, transfer the cells to a 1.5 ml tube.
  7. Count the cells using 10 µl cell suspension, 10 µl trypan blue and the Neubauer chamber.
  8. Transfer 5 x 104 cells to a new 1.5 ml tube.
  9. Centrifuge the cell suspension 5 min with 300 x g at room temperature.
  10. Remove and discard the supernatant by pipetting and add 300 µl serum-free medium, pipette up and down to resolve cell pellet.
  11. Take a new 12 well plate and add 500 µl of FBS containing full growth medium in each well.
  12. Add a ThinCertsTM to each 12 well.
  13. Transfer the complete cell suspension from step 10 onto a ThinCertTM.
  14. Let the cells migrate for 24-48 h at 37 °C in the CO2 incubator.
  15. Prepare a new 12 well plate with 1 ml 1x sterile PBS in each well.
  16. Prepare a new 12 well plate with 500 µl accutase solution in each well.
  17. Remove the serum-free medium in the ThinCertsTM using a pipette.
  18. Wash the cells first by transferring the ThinCertsTM to the plate with PBS to remove remaining medium and transfer the ThinCertsTM directly to the plate containing accutase.
  19. Incubate the cells in the incubator to detach them. Extend the cell specific detaching time from step 5 for additional 5 min.
  20. Gently tap the ThinCertsTM against the wall of the well to ensure the complete detachment of the cells; discard the ThinCertsTM.
  21. Add 1 ml FBS containing full growth medium to the well and transfer cells to a 1.5 ml tube.
  22. Centrifuge the cell suspension 5 min with 300 x g at room temperature.
  23. Gently remove the supernatant with a pipette and resuspend the cells with 1 ml PBS.
  24. Centrifuge the cell suspension 5 min with 300 x g at room temperature.
  25. Gently remove the supernatant, add 100 µl of 1x cell lysis buffer, vortex for 5 sec and incubate for 10 min at room temperature.
  26. Pellet the cells for 3 min with 13,000 x g at room temperature.
  27. In the meantime, prepare the LDH reaction mixture (according to the cytotoxicity detection kit manufacturer’s protocol).
  28. Transfer 40 µl of the cell supernatant into a 96 well plate using two technical replicates.
  29. Add 100 µl of the reaction mixture and incubate 10-30 min at room temperature. Protect the plate from light.
  30. Measure the relative LDH activity at 490 nm. The reference wavelength should be more than 600 nm.

Notes

  1. The migration of cell lines can vary greatly. An initial test without a treatment of the cells might be helpful, especially when different cell lines are used.
  2. The incubation time with the reaction mixture can be increased up to 1 h, if necessary.
  3. The optical density (OD490) should reach at least 0.1 to ensure a valid enzyme activity. Lower values indicate a poor migration, since some cell lines do not migrate through the ThinCertsTM (e.g., the melanoma cell line MeWo).

Acknowledgments

We would like to thank The Federal Ministry of Education and Research, Germany and the German Center for Lung Research who financially supported this study.

References

  1. Schneider, M. A., Granzow, M., Warth, A., Schnabel, P. A., Thomas, M., Herth, F. J., Dienemann, H., Muley, T. and Meister, M. (2015). Glycodelin: A new biomarker with immunomodulatory functions in non-small cell lung cancer. Clin Cancer Res 21(15): 3529-3540.

简介

肿瘤细胞的高迁移率通常导致患者的生存的不良预后。 在这里,我们描述了使用定量测定测量细胞迁移的协议。 使用ThinCerts TM细胞培养插入物和乳酸脱氢酶(LDH)测定来测量相对肿瘤细胞迁移以定量相对细胞数。 使用LDH试剂盒的迁移的定量比使用结晶蓝计数细胞的其它方法精确得多。

关键字:迁移实验, Transwells, 肿瘤细胞, LDH法

材料和试剂

  1. 12孔细胞培养板(Greiner Bio One International GmbH,目录号:665180)
  2. 96孔组织培养试验板(TPP Techno Plastic Products AG,目录号:92696)
  3. 1.5ml管(Carl Roth GmbH + Co.,目录号:7080.1)
  4. 细孔直径为8μm的ThinCerts TM 细胞培养插入物(Greiner Bio-One GmbH,目录号:665638)
  5. 用于血球计的盖玻片(Carl Roth GmbH + Co.,目录号:L189.1)
  6. 过滤嘴(10μl,20μl,100μl,200μl和1000μl)(Carl Roth GmbH + Co.,目录号:771288,774288,772288,739288和740288)
  7. 肿瘤细胞(肺癌细胞系H1975和2106T)
  8. 细胞培养基(细胞系特异性),有和没有胎牛血清(FBS)
  9. 热灭活的FBS(E.U.-批准,南美来源)(Thermo Fischer Scientific,目录号:10500-064)
  10. 1×Dulbecco磷酸盐缓冲盐水(DPBS,无钙,无镁)(Thermo Fischer Scientific,Gibco TM,目录号:14190-094)
  11. (Thermo Fischer Scientific,Gibco TM ,目录号:A11105-01),其中所述试剂盒包括:
  12. 丝裂霉素C(AppliChem GmbH,目录号:A2190,0002)
  13. 台盼蓝溶液(Sigma-Aldrich,目录号:T8154)
  14. 10x细胞裂解缓冲液(Cell Signaling Technology,目录号:9803S)
  15. 细胞毒性检测试剂盒(LDH)(Roche Diagnostics,目录号:11644793001)

设备

  1. CO 2细胞培养箱(Panasonic Corporation,Sanyo,型号:SANYO-InCu saFe )
  2. Neubauer细胞计数室(VWR International,目录号:BRND717810)
  3. 微量离心机(Eppendorf AG,型号:5417R)
  4. 涡旋混合器(VWR International,目录号:444-1372)
  5. 多功能微孔板吸光度读数器(Tecan Trading AG,型号:日出 TM
  6. Gilson's Pipetman经典移液器(P10,P20,P100,P200,P1000)

程序


图1. 体外肿瘤细胞迁移测定的流程图

  1. 在12孔细胞培养板中用期望的测定(例如,靶基因的siRNA敲除或药物递送)处理细胞。
  2. 在37℃下,在CO 2培养箱中,将生长培养基更换1ml完全无血清培养基16小时过夜。
  3. 在37℃在CO 2培养箱中,用1ml无血清培养基中的10μg/ml丝裂霉素C处理细胞2小时,以抑制细胞增殖。
  4. 用1ml无菌1x PBS洗涤细胞一次
  5. 加入250微升accutase溶液,孵育细胞约5-10分钟(取决于所使用的细胞的粘附),以分离它们。
  6. 向细胞中加入1ml无血清培养基,将细胞转移到1.5ml管中
  7. 使用10微升细胞悬液,10微升台盼蓝和Neubauer室计数细胞
  8. 将5×10 4个细胞转移到新的1.5ml管中
  9. 在室温下用300×g离心细胞悬浮液5分钟。
  10. 通过吸取除去和丢弃上清液,加入300μl无血清培养基,上下吸取以解决细胞沉淀。
  11. 取一个新的12孔板,并在每个孔中加入500μl含有完全生长培养基的FBS
  12. 向每个井中添加ThinCerts TM
  13. 将第10步中的完整细胞悬液转移到ThinCert TM 上。
  14. 让细胞在CO 2培养箱中在37℃下迁移24-48小时。
  15. 准备一个新的12孔板与1毫升1×无菌PBS在每个孔
  16. 准备一个新的12孔板与500微升accutase溶液在每个孔
  17. 使用移液器移除ThinCerts TM 中的无血清培养基。
  18. 先用ThinCerts TM 清洗细胞,用PBS清洗剩余的培养基并转移ThinCerts TM 直接到含有accutase的板
  19. 孵育细胞在孵化器中分离他们。 将细胞特异性分离时间从步骤5延长另外5分钟。
  20. 轻轻敲打ThinCerts TM ,以确保细胞完全分离; 舍弃ThinCerts TM
  21. 加入1ml含有完全生长培养基的FBS到孔中,并将细胞转移到1.5ml管
  22. 在室温下用300×g离心细胞悬浮液5分钟。
  23. 用移液管轻轻取出上清液,用1ml PBS重悬细胞
  24. 在室温下用300×g离心细胞悬浮液5分钟。
  25. 轻轻除去上清液,加入100μl的1x细胞裂解缓冲液,涡旋5秒,并在室温下孵育10分钟。
  26. 在室温下用13,000×g的细胞沉淀细胞3分钟
  27. 同时,制备LDH反应混合物(根据细胞毒性检测试剂盒制造商的方案)
  28. 使用两个技术重复转移40微升的细胞上清液到96孔板。
  29. 加入100μl反应混合物,并在室温下孵育10-30分钟。 保护板免受光照。
  30. 测量490nm处的相对LDH活性。 参考波长应大于600nm。

笔记

  1. 细胞系的迁移可以极大地变化。 没有处理细胞的初始测试可能是有帮助的,特别是当使用不同的细胞系时。
  2. 如果需要,与反应混合物的孵育时间可以增加至1小时
  3. 光密度(OD 490)应该达到至少0.1以确保有效的酶活性。 较低的值表示迁移不良,因为一些细胞系不通过ThinCerts TM迁移 /em>,黑色素瘤细胞系MeWo)

致谢

我们要感谢联邦教育和研究部,德国和德国肺研究中心,他们在财政上支持这项研究。

参考文献

  1. Schneider,M.A.,Granzow,M.,Warth,A.,Schnabel,P.A.,Thomas,M.,Herth,F.J.,Dienemann,H.,Muley,T.and Meister,M.(2015)。 Glycodelin:一种在非小细胞肺癌中具有免疫调节功能的新生物标记物。 Clin Cancer Res 21(15):3529-3540。
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引用:Schneider, M. A. (2016). In vitro Tumor Cell Migration Assay Using ThinCertsTM (Transwells). Bio-protocol 6(11): e1830. DOI: 10.21769/BioProtoc.1830.
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