Quanto è utile/interessante questa discussione:
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gigim
Nuovo Arrivato
6 Messaggi |
 Inserito il - 13 settembre 2010 : 10:32:48
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Salve a tutti, sto cercando qualche buon protocollo per l'isolamento e successiva messa in coltura di fibroblasti, da tessuti prelevati da carcinoma mammario. Grazie anticipatamente a tutti coloro mi possono essere d'aiuto, ciao ciao
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gigim
Nuovo Arrivato
6 Messaggi |
Inserito il - 13 settembre 2010 : 13:45:49
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Tra i protocolli esaminati questo mi sembra il più affidabile.
Generation of primary cell lines.
DA: (Expression of B7-H1 in breast cancer patients is strongly associated with high proliferative Ki-67-expressing tumor cells. Hazem Ghebeh et al.)
Tissues obtained from breast cancer patients and new born baby foreskin were placed in DMEM medium (Sigma, Saint Louis, MO) supplemented with 10% fetal calf serum (FCS) (Cambrex, Rockland, ME) and kept at 4 C until processed. Tissue were cut into smaller pieces (1–2 mm diameter), placed in digestion solution (400 IU collagenase, 100 IU hyaluridinase and 10% serum, all from Sigma, Saint Louis, MO), containing antibiotics and antimycotics solution (Gibco, Grand Island, NY) and incubated at 37 C for 6 hr to overnight in an Adams Nutator Mixer (Becton Dickinson, Franklin Lakes, NJ). The cells were separated by differential centrifugation at 90g for 2 min. The epithelial cells in the pellet were cultured at 37 C, 5% CO2 in 75 cm2 T-flask containing 12 mL of epithelial cell growth medium composed of Medium 199 and Ham’s F12 Media mixed 1:1 and supplemented with 10 lg/mL Insulin, 5 lg/mL transferrin, 250 ng/mL Hydrocortisone, 6.1 lg/mL Ethanolamine, 14.1 lg/mL O-phosphoethanolamine, 8.8 lg/mL Prostaglandin E1, 10 ng/mL epidermal growth factor (EGF), 40 lg/mL Fetuin, 100 lg/mL bovine serum albumin (All from Sigma, Saint Louis, MO) and 20 mg bovine pitutiary extract (GIBCO, Grand Island, NY). The supernatant containing fibroblasto were centrifuged at 485g for 8 min, resuspended in fibroblasts growth medium (Medium 199 and Ham’s F12 mixed 1:1 and supplemented with 10–20% FCS) and cultured at 37 C, 5% CO2. Skin epithelial cells (keratinocytes) were cultured in keratinocyte serum-free medium (K-SFM) (GIBCO, Grand Island, NY).
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Fibroblasti da tessuto
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