Kallyne,

Ideally you should stardardize the amount of tissue by concentration of total protein or mg of tissue (weight). In this way, your results will determine the concentration of cytokine per mg of tissue (e.g. pg of IFN-g/100 mg of skin) or per ug of total protein (pg of IFN-g/ug of total protein).

Usually we work with tissue homogenates from liver, spleen and lungs but it may work as well for skin. Please let me know if you need any protocol.

Hi Kallyne, I think if you standardize the sample protein is fine but is not necessary , also you can dilute the samples according to the ELISA kit protocol. since the ELISA assay containing a standard curve to calculate the levels of cytokines ...

Kallyne,

Ideally you should stardardize the amount of tissue by concentration of total protein or mg of tissue (weight). In this way, your results will determine the concentration of cytokine per mg of tissue (e.g. pg of IFN-g/100 mg of skin) or per ug of total protein (pg of IFN-g/ug of total protein).

Usually we work with tissue homogenates from liver, spleen and lungs but it may work as well for skin. Please let me know if you need any protocol.

Thank you very much for the answers Huan Hu and Ricardo.

Ricardo , I really appreciate if you send me the protocol to homogenate your samples. I also believe that your protocol may work as well for skin.

Hello

i agree with Ricardo Toshio Fujiwara ,you can use weigh your tissue samples before homogenization if this is not possible then the next best thing is to use protein concentration , ore take a specific volume of homogenate . For example,if you take 100ul of the homogenate for cytokine estimation,you can take another 100ul for protein estimation . Do it in duplicates or triplicates and represent at pg/ug or ng/mg of the cytokine/protein.

in our lab followed this protocol for Skin Tissue Homogenization Procedures by ELISA :

1. Skin punches measuring 6 mm were homogenized in 1.5 mL extraction buffer (containing 10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100) per gram of tissue using a glass homogenizer. The homogenates then transferre to 1.5 mL Eppendorf tubes, centrifuged at 13,000 xg for 10 minutes at 4°C, and the supernatant was stored at -80°C until analyzed. TNF-alpha and IL-1beta protein levels were determined using KRC3012 and KRC0012.

2. Skin flap biopsies obtaine at different times after grafting and immediately stored under frozen nitrogen.On the day of the assy, the biopsies were allows to thaw and 20-50 mg of wet

tissue was homgenized with the aid of a Polytron homogenizer in ice-cold PBS containing a protease-inhibitor cocktail. The homogenates were centrifuged for 20 minutes at 10,000 x g to remove debris and insoluble material, and aliquots of the supernatants were assayed for total protein content by the BCA method and rat interferon-gamma was assayed by ELISA.

Reference:

1- Blalock, T.D., J.C. Varela, S. Gowda, Y. Tang, C. Chen, B.A. Mast, and G.S. Schultz (2001) Ischemic skin wound healing models in rats. Wounds 13(1):35-44.

2- Fernandez-Botran, R., V. Gorantla, X.C. Sun, X.P. Ren, G. Perez-Abadia, F.A. Crespo,R. Oliver, H.I. Orhu, E.E. Quan, C. Maldonado, M. Ray, and J.H. Barker (2002) Targeting of glycosaminoglycan-cytokine interactions as a novel therapeutic approach in allotransplantation.Transplantation 74(5):623-629 (cites the use of KRC4022 with skin biopsies).

i hope help you , good luck

Thanks a lot for the information Saleh Alkarim. It was very helpfull!