Materials and Reagents

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

Equipment

Homogenizer such as Precellys (Ozyme BER1011S, France) or ultraturax
96 wells plates (DUTSCHER SCIENTIFIC, catalog number: 047632 )
Luminoskan (Thermo Fisher Scientific, catalog number: 5210470 )
Centrifuges

Procedure

20 mg of fresh or frozen tumor tissues were resuspended in 200 μl of extraction buffer, on ice. The extaction buffer contains protease inhibitors.
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.
The homogenate was centrifuged for 10 min at 6,000 rpm, at 4 °C.
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.
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.
In parallel, resuspend hemoglobin (concentration of stock solution is 10 mg/ml) in the extraction buffer.
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.
Add 10 μl of sample or standards per well in triplicate.
Add 100 μl of Drabkin’s reagent/Brij 35 solution 30%.
Incubate 15 min at room temperature. The reaction is stable a couple of hours.
Determine the optical density of each well, using a microplate reader set to 540 nm.
Average the triplicate readings for each standard, control, and sample and subtract the average zero standard optical density.
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.
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

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.