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Measurement of Hemoglobin
血红蛋白检测   

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Abstract

This protocol allows to measure the levels of intratumoral hemoglobin from human or rodent fresh samples but also frozen tumors. The advantage of this method is to use very few microliters of biological material for hemoglobin and the protocol is carried out quickly.

Keywords: Cancer(癌症), Angiogenesis(血管生成), Resistance to treatment(治疗抵抗)

Materials and Reagents

  1. Fresh or frozen tumor tissues
  2. Extraction buffer (Life Technologies, catalog number: FNN0011 )
  3. Human hemoglobin (Sigma-Aldrich, catalog number: H7379 )
  4. Drabkin's reagent (Sigma-Aldrich, catalog number: D5941 )
  5. Brij 35 Solution 30% (Sigma-Aldrich, catalog number: B4184 )
  6. Antifoam Y-30 Emulsion (Sigma-Aldrich, catalog number: A5758 )
  7. BCA protein quantification kit (Interchim, catalog number: MP2920 )
  8. Extaction buffer

Equipment

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

Procedure

  1. 20 mg of fresh or frozen tumor tissues were resuspended in 200 μl of extraction buffer, on ice. The extaction buffer contains protease inhibitors.
  2. Tissues were mechanically ground using a homogenizer such as an ultraturax or a Precellys. With ultraturax an antifoam solution (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. Reconstitute a vial of Drabkin's reagent in 1 L of water, add 0.5 ml of Brij 35 solution 30%. This solution can be stored at room temperature for 6 months, protected from light.
  6. In parallel, resuspend hemoglobin (concentration of stock solution is 10 mg/ml) in the extraction buffer.
  7. Achieve an eight point standard curve using 2-fold serial dilutions of hemoglobin, in extraction buffer, and a high standard of 2,000 pg/μl is recommended. To obtain the concentration of 2,000 pg/μl, the working solution (10 mg/ml) must be diluted 5 times.
  8. Add 10 μl of sample or standards per well in triplicate.
  9. Add 100 μl of Drabkin’s reagent/Brij 35 solution 30%.
  10. Incubate 15 min at room temperature. The reaction is stable a couple of hours.
  11. Determine the optical density of each well, using a microplate reader set to 540 nm.
  12. Average the triplicate readings for each standard, control, and sample and subtract the average zero standard optical density.
  13. 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.
  14. Hemoglobin concentration of samples must be divided by the protein concentration determined by BCA to obtain a result in microgram per micro liter per microgram of protein.

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.

简介

该协议允许测量来自人或啮齿动物新鲜样品的肿瘤内血红蛋白的水平,但也测量冷冻肿瘤的水平。 该方法的优点是使用非常少的微生物材料用于血红蛋白,并且该方案快速进行。

关键字:癌症, 血管生成, 治疗抵抗

材料和试剂

  1. 新鲜或冷冻的肿瘤组织
  2. 提取缓冲液(Life Technologies,目录号:FNN0011)
  3. 人血红蛋白(Sigma-Aldrich,目录号:H7379)
  4. Drabkin试剂(Sigma-Aldrich,目录号:D5941)
  5. Brij 35 Solution 30%(Sigma-Aldrich,目录号:B4184)
  6. 消泡剂Y-30乳液(Sigma-Aldrich,目录号:A5758)
  7. BCA蛋白定量试剂盒(Interchim,目录号:MP2920)
  8. 提取缓冲区

设备

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

程序

  1. 将20mg新鲜或冷冻的肿瘤组织在冰上重悬浮于200μl提取缓冲液中。 脱离缓冲液含有蛋白酶抑制剂
  2. 使用均化器如ultraturax或Precellys对组织进行机械研磨。 使用ultraturax,使用消泡剂溶液(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. 在1L水中重构一瓶Drabkin's试剂,加入0.5ml 30%的Brij 35溶液。 该溶液可在室温下储存6个月,避光
  6. 同时,在提取缓冲液中重悬血红蛋白(储备溶液的浓度为10mg/ml)
  7. 使用2倍系列稀释的血红蛋白,在提取缓冲液中,并建议高标准的2,000 pg /μl达到八点标准曲线。 为了获得2,000pg /μl的浓度,工作溶液(10mg/ml)必须稀释5倍
  8. 每孔加入10μl样品或标准品一式三份
  9. 加入100μlDrabkin试剂/Brij 35溶液30%
  10. 在室温下孵育15分钟。 反应稳定几小时。
  11. 使用设置为540nm的酶标仪测定每个孔的光密度
  12. 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
  13. 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。
  14. 样品的血红蛋白浓度必须除以由BCA测定的蛋白质浓度,以获得微克/微升/微克蛋白质的结果。

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:合同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",罗氏法国和"协会 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/阿瓦斯丁治疗后加速小鼠中透明细胞肾细胞癌生长:CXCL细胞因子的作用 。 Oncogene 31(13):1683-1694。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Grépin, R. and Pagès, G. (2012). Measurement of Hemoglobin. Bio-protocol 2(22): e291. DOI: 10.21769/BioProtoc.291.
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Paul Diaz
Cyternity, Inc.
I'm planning on adopting this protocol to assay %hemolysis in blood samples. What has been your limit of detection?
11/5/2013 6:21:56 PM Reply