Hi, I am working with 3D organotypic co-cultures (skin equivalents), using human keratinocytes and fibroblasts. I can section these cultures and make immunflourescene stainings with antibodies but never tried to make protein lysates from them. If any one did this or have been successful in doing so, please share your tips or protocol. thank you so much in advance.
Your question it´s a little bit tricky.
If you want to have easy cell lysates from the overall culture, you can colelct them (by trypsin or with a cell scraper), resuspending them in a lysis buffer (e.g. RIPA buffer) and analysing the protein content in WB.
According to the desired proteins you want to see, several different lysis buffer can be used: nuclear and mitochondrial proteins can be efficiently extracted with RIPA buffer; for cytoplasm proteins I would go for an easy 0.5-1% NP40 buffer (in 50mM tris pH7.4, 150mM NaCl, protease inhibitors, ecc...)
Bye
Depending on the purposes of following experiment. My colleague made it works in Immunoprecipitation and Kinase assay. Please see the reference paper Noya et al., 2001. J. Virol. July 2001 vol. 75 no. 13 6121-6134. doi: 10.1128/JVI.75.13.6121-6134.2001
Article p21cip1 Degradation in Differentiated Keratinocytes Is Abrog...
Presumably, you want to analyze the proteins in the dermal and epidermal equivalent of your organotypic co-cultures separately. What worked very well in our hands was incubation of the 3D culture (excised from the filter insert) in buffered 10 mM dithiothreitol/DTT for 1h at 4° C. You can peel off the epithelium from the collagen gel (carefully!) and prepare lysates/extracts separately. In well developed differentiated cultures there is a pretty clean separation within the basement membrane, proteins remain intact, and the viability of the cells is preserved (no major damage). An important advantage for the epidermal part is the removal of the voluminous collagen mass. For details see: J Cell Sci 117, 2611-22, 2004 and J Invest Dermatol 127, 545-54, 2007 (current e-mail, [email protected]). Good luck.
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