We have use two types of isolation protocols:

The first protocol was developed for the isolation of tumor tissue. After the enucleation the tumor was removed from the bulbus, half of it was taken for histological examination, the other half of the tumor was used for cell isolation. The tumor was disinfected once with 70% ethanol and four times with Betadine solution. For each step new equipment was used. After the disinfection the tissue was minced to 2x2 mm pieces with 20 gauge hypodermic needle, and digested in 400 U/ml collagenase IV and 0,25% trypsin/EDTA in Ham’s F12 media. The digestion lasted 60 minutes. After the 60 minutes the cell suspension was centrifuged on 500G, 5 min, then the cell viability was examined directly with a Bürker chamber using trypan blue stain. The cells were moved into a 25cm2 cell culture flask into Ham’s F12 medium supplemented by 10% foetal bovine serum (FBS) and 1% penicillin neomycin streptomycin solution (PSN). After 24 hours the media were remowed, and only the adherent cells were cultivated.

The second isolation protocol was developed for fine needle aspiration biopsy. The cell suspension was centrifuged and after viability assay, the cells were cultured in a 25 cm2 cell culture flask in RPMI-1640 medium supplemented by 10% foetal bovine serum (FBS) and 1% penicillin streptonycin neomycin (PSN) solution. After 24 hours the media was removed and only the adherent cells were cultured.


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