Transplanted cells ben about 24 hours CONFIRMS to recover and return to growth. MCF-7 cell lines MCF-7 human breast adenocarcinoma cells originally isolated from a woman 69 years old caucasian with several characteristics of differentiated mammary epithelium from the American Type Culture Collection as passage 146 or more dd and obtained grown iNititally 1,500 cells/cm2 in DMEMmedium, 10% heat-inactivated f Fetal K Calf serum, 2 mM L-glutamine, 1 mM Na pyruvate and 1 mM penicillin STF-62247 STF62247 / streptomycin. MDA MB 231 human cell lines MDA MB 231 breast adenocarcinoma glands as part of a series of tumor cell lines of breast pleural effusions of a Caucasian woman of 47 years  and cultured inititally to get 1500 cells/cm2 in Leibovitz L 15 medium with 10% FCS, 2 mM L-glutamine and 1 mM penicillin / streptomycin. Electron microscopy of mammary tissues were on Objekttr Willingly cultivated for scanning and transmission electron microscopy are. After the ex vivo growth of tumor cells from different cultures have been obtained on the Objekttr hunter in a L Solution, the fixed 3% glutaraldehyde in 0. 1 M sodium cacodylate, pH 7 4 for at least 24 hours.
Then the samples in 1% OsO4 in H2O contribution, dehydrated in a graded ethanol before. SEM for the critical point drying, the samples with gold and palladium 35CF SSM were coated examined in a scanning electron microscope JEOL at 15 kV. For TEM, ethanol containing dried breast Belinostat tumor tissue in Epon. Ultrathin sections were found with uranyl acetate and lead acetate Rbt and examined under a microscope Philips CM10 electronics at 80 kV. Immunofluorescence mammary tumor derived cells were Objekttr Willingly bred × 3 with PBS / Tween 20 for 5 min and air-dried for 60 min. Subsequently End, the samples were fixed with cold acetone for 10 minutes, and rehydrated in PBS for 5 min.
After treatment with PBS / 5% BSA for 10 minutes to block nonspecific binding sites, the samples were labeled with mouse anti-vimentin, Dako, Hamburg, Germany was incubated 30 min. Min after three washes with PBS / Tween 20 for 5 each, the samples were labeled with mouse anti TRITClabelled secondary Ren Dako for incubated for 90 min. 3 more W between With PBS / Tween 20 were carried out for 5 minutes, and after blocking with mouse serum, Dako, samples were incubated with FITC-conjugated monoclonal Body anti pan cytokeratin, Dako incubated for 90 min. Min after three washes with PBS / Tween 20 for 5, the samples were incubated with medium with DAPI, to obtain at the same time. Samples for subsequent immunofluorescence microscopy Were derived for the background color and embroidered on the tumor cells of passages with sera from M The corresponding IgG subclass nozzles instead of prim Ren antique Incubated rpern.
Fluorescence microscopy was performed with an Olympus E SIS II CCD camera see YEARS Performed engined fluorescence microscope Olympus IX 50th The fluorescence image analysis and superposition of fluorescence was obtained with the analysis beam SIS software image B. As a result, cytokeratin filaments showed green, red filaments of vimentin and DNA in the nuclei of cells blue fluorescence. Cytokeratin and vimentin quantification by flow cytometry were fixed approximately 5105 × breast tumor-derived cells by successive addition of ice-cold ethanol to a final concentration of 70%. Subsequently End, the cells were stored at 4 for at least 24 h.