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This protocol allows to measure the levels cytokines - such as VEGFs, CXCLs cytokines, PDGF or FGF - from fresh samples but also frozen tumors. The advantage of this method is to use very few micrograms of biological material and the protocol is carried out quickly.

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Measurement of Cytokines
细胞因子检测

癌症生物学 > 通用技术 > 生物化学试验 > 蛋白质分析
作者: Renaud Grépin
Renaud GrépinAffiliation: Medical Biology Division, Centre Scientifique de Monaco, Monaco, Monaco
Bio-protocol author page: a161
 and Gilles Pagès
Gilles PagèsAffiliation: Institute for Research on Cancer and Aging of Nice (IRCAN) UMR CNRS 7284/U INSERM 1081, University Nice Sophia Antipolis, Nice, France
For correspondence: gpages@unice.fr
Bio-protocol author page: a162
Vol 2, Iss 22, 11/20/2012, 5094 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.290

[Abstract] This protocol allows to measure the levels cytokines - such as VEGFs, CXCLs cytokines, PDGF or FGF - from fresh samples but also frozen tumors. The advantage of this method is to use very few micrograms of biological material and the protocol is carried out quickly.
Keywords: Cytokines(细胞因子), ELISA(ELISA), Frozen tumors(冷冻的肿瘤), Fresh samples(新鲜样品)

[Abstract] 该实验方案的中文版正在准备中...

Materials and Reagents

  1. Fresh or frozen tumor tissues stored at - 80 °C
  2. Extraction buffer (Life Technologies, catalog number: FNN0011 )
  3. Antifoam Y-30 Emulsion (Sigma-Aldrich, catalog number: A5758 )
  4. BCA protein quantification kit (Interchim, catalog number: MP2920 )
  5. ELISA kits (Peprotech or R&D System)
  6. Tween-20 (Sigma-Aldrich, catalog number: P-7949 )
  7. BSA (Sigma-Aldrich, catalog number: A-7030 )
  8. Avidin-HRP conjugate solution (Sigma-Aldrich, catalog number: A-7419 )
  9. 10x Dulbecco’s PBS (Life Technologies, Gibco®, catalog number: 14200-075 )
  10. ABTS Liquid substrate solution (Sigma-Aldrich, catalog number: A3219 )

Equipment

  1. Homogenizer such as Precellys (Ozyme BER1011S, France) or ultraturax
  2. Centrifuge
  3. 96 wells plates (DUTSCHER SCIENTIFIC, catalog number: 0 47632 )
  4. ELISA microplates (Nunc MaxiSorp, catalog number: 439454 )
  5. Luminoskan (Thermo Fisher Scientific, catalog number: 5210470 )

Procedures

  1. 20 mg of fresh or frozen tumor tissues were resuspended in 200 μl of extraction buffer at 4 °C.
  2. Tissues were mechanically ground using a homogenizer such as an ultraturax or a Precellys. With ultraturax an antifoam solution as described by the manufacturer (Sigma-Aldrich – antifoam Y-30 Emulsion) is used.
  3. The homogenate was centrifuged for 10 min at 6,000 rpm, at 4 °C.
  4. The supernatant was recovered. 10 μl supernatant is used to determinate the protein concentration using a protein assay such as BCA. The sample can be stored at -80 °C.
  5. Measurement of cytokines must be performed as described by ELISA kit manufacturer.
  6. Dilute capture antibody with PBS to a concentration of 1 μg/ml and add 100 μl to each ELISA pate well. Seal the plate and incubate overnight at room temperature.
  7. Invert plate to remove capture antibody and blot on paper towel and wash plate 3 times by adding 300 μl of wash solution (1x PBS – 0.05% tween-20). Invert plate to remove wash solution and blot on paper towel.
  8. Block plate by adding 300 μl per well of 1% BSA in 1x PBS. Incubate 1 h at room temperature.
  9. Invert plate to remove blocking buffer and wash plate 3 times as described in step 7.
  10. Achieve an eight point standard curve using 2-fold serial dilutions of standard, in extraction diluent solution (1x PBS – 0.05% Tween-20 – 0.1% BSA), and a high standard of 2,000 μg/μl is recommended.
  11. Add 100 μl of standard or sample to each well in triplicate. Incubate at room temperature for at least 2 h.
  12. Invert plate to remove samples and standard and wash plate 3 times as described in step 7.
  13. Dilute biotinylated detection antibody in diluent (1x PBS - 0.05% Tween-20 - 0.1% BSA) to a concentration of 500 ng/ml and add 100 μl per well. Incubate at room temperature for 2 h.
  14. Invert plate to remove detection antibody and wash plate 3 times as described in step 7.
  15. Add 100 μl per well of Avidin-HRP conjugate diluted 1: 2,000 in diluent (1x PBS – 0.05% tween-20 - 0.1% BSA). Incubate 30 min at room temperature.
  16. Invert plate to remove Avidin-HRP conjugate and wash plate 3 times as described in step 7.
  17. Add 100 μl of ABTS liquid subtrate to each well. Incubate at room temperature, 10 min, for color development.
  18. Determine the optical density of each well, using a microplate reader set to 405 nm.
  19. Average the triplicate readings for each standard, control, and sample and subtract the average zero standard optical density.
  20. Create a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a curve through the points on the graph.

Acknowledgments

Our protocol was adapted from the following article: Grepin et al. (2012). We thank Dr Brahimi-Horn for editorial assistance. Financial support: Contract VEGFIL from the National Institute of Cancer (INCA), the French Association for Cancer Research (ARC), the Foundation de France, the “Conseil Général des Alpes Maritimes”, Roche France and The “Association pour la Recherche sur les Tumeurs du Rein (ARTuR)”.

References

  1. Grepin, R., Guyot, M., Jacquin, M., Durivault, J., Chamorey, E., Sudaka, A., Serdjebi, C., Lacarelle, B., Scoazec, J. Y., Negrier, S., Simonnet, H. and Pages, G. (2012). Acceleration of clear cell renal cell carcinoma growth in mice following bevacizumab/Avastin treatment: the role of CXCL cytokines. Oncogene 31(13): 1683-1694.

材料和试剂

  1. 新鲜或冷冻的肿瘤组织贮存在-80℃下
  2. 提取缓冲液(Life Technologies,目录号:FNN0011)
  3. 消泡剂Y-30乳液(Sigma-Aldrich,目录号:A5758)
  4. BCA蛋白定量试剂盒(Interchim,目录号:MP2920)
  5. ELISA试剂盒(Peprotech或R& D系统)
  6. 吐温-20(Sigma-Aldrich,目录号:P-7949)
  7. BSA(Sigma-Aldrich,目录号:A-7030)
  8. 抗生物素蛋白-HRP偶联物溶液(Sigma-Aldrich,目录号:A-7419)
  9. 10x Dulbecco's PBS(Life Technologies,Gibco ,目录号:14200-075)
  10. ABTS液体底物溶液(Sigma-Aldrich,目录号:A3219)

设备

  1. 均化器如Precellys(Ozyme BER1011S,France)或ultraturax
  2. 离心机
  3. 96孔板(DUTSCHER SCIENTIFIC,目录号:047632)
  4. ELISA微孔板(Nunc MaxiSorp,目录号:439454)
  5. Luminoskan(Thermo Fisher Scientific,目录号:5210470)

程序

  1. 将20mg新鲜或冷冻的肿瘤组织重悬于4℃的200μl提取缓冲液中
  2. 使用均化器如ultraturax或Precellys对组织进行机械研磨。 使用ultraturax,使用如制造商描述的消泡溶液(Sigma-Aldrich-消泡剂Y-30乳液)。
  3. 将匀浆在4℃下以6,000rpm离心10分钟
  4. 回收上清液。 10μl上清液用于使用蛋白质测定法如BCA测定蛋白质浓度。 样品可以储存在-80℃
  5. 细胞因子的测量必须如ELISA试剂盒制造商所述进行。
  6. 用PBS稀释捕获抗体至浓度为1μg/ml,并向每个ELISA板中加入100μl。 密封板并在室温下孵育过夜
  7. 通过加入300μl洗涤溶液(1×PBS-0.05%tween-20)反转板以除去捕获抗体并在纸巾和洗涤板上印迹3次。 倒置板,以除去洗涤溶液,并在纸巾上吸干
  8. 通过加入300μl/孔的1%BSA的1%PBS封闭平板。 在室温下孵育1小时。
  9. 按照步骤7所述,颠倒板以去除封闭缓冲液并洗涤板3次。
  10. 使用2倍系列稀释的标准品在提取稀释液(1×PBS-0.05%Tween-20 - 0.1%BSA)中达到8点标准曲线,推荐使用高标准的2,000μg/
  11. 每孔加入100μl标准品或样品一式三份。 在室温下孵育至少2小时。
  12. 按照步骤7所述,翻转平板以取出样品,并按标准和洗板重复3次。
  13. 稀释生物素化的检测抗体稀释液(1×PBS - 0.05%吐温-20 - 0.1%BSA)至500 ng/ml的浓度,每孔加入100μl。 在室温下孵育2小时
  14. 按照步骤7所述,颠倒平板以去除检测抗体并洗涤板3次。
  15. 在稀释液(1×PBS-0.05%吐温-20-0.1%BSA)中以1:2000稀释的亲和素-HRP缀合物加入100μl/孔。 在室温下孵育30分钟。
  16. 按照步骤7中所述颠倒平板以去除抗生物素蛋白-HRP缀合物和洗板3次
  17. 向每个孔中加入100μlABTS液体底物。 在室温下孵育10分钟,用于显色
  18. 使用设置为405nm的酶标仪测定每孔的光密度。
  19. 平均每个标准,对照和样品的三次读数,并减去平均零标准光密度
  20. 通过绘制y轴上每个标准物的平均吸光度与x轴上的浓度并绘制通过图上的点的曲线来创建标准曲线。

致谢

我们的方案改编自以下文章:Grepin等人(2012)。我们感谢Brahimi-Horn博士提供编辑协助。财务支持:来自国家癌症研究所(INCA),法国癌症研究协会(ARC),法国基金会,"法国海滨旅游局",罗氏法国的合同VEGFIL和" Tumurs du Rein(ARTuR)"。

参考文献

  1. Greene,R.,Guyot,M.,Jacquin,M.,Durivault,J.,Chamorey,E.,Sudaka,A.,Serdjebi,C.,Lacarelle,B.,Scoazec,JY,Negrier, ,H.和Pages,G。(2012)。 在贝伐珠单抗/阿瓦斯丁治疗后小鼠中透明细胞肾细胞癌生长的加速:CXCL细胞因子的作用 Oncogene 31(13):1683-1694。
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How to cite this protocol: Grépin, R. and Pagès, G. (2012). Measurement of Cytokines. Bio-protocol 2(22): e290. DOI: 10.21769/BioProtoc.290; Full Text



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2/9/2013 2:00:05 AM  

Akinwande Kazeem
FEDERAL MEDICAL CENTRE ABEOKUTA

Please how can one acquire reagents for this method of extraction? How accessible are they?

2/14/2013 11:33:02 PM  

Fanglian He
Department of Biology, University of Pennsylvania, USA

Authors have provided the catalog numbers for most of reagents used in this protocol. So, you have to check with suppliers about the availability.

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11/16/2012 5:55:08 AM  

The voice of rtaoinlaity! Good to hear from you.

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Renaud Grépin的其他实验方案(1)
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