on the Lebensf ability of the cells EGCG was also GDC-0879 studied. Materials. HT29, a line of c Lon human adenocarcinoma cells and its subclone mucus HT29 MTX E12 were obtained by courtesy of Thomas Kissel. Dulbecco’s modified Eagle, s medium with Glutamax, 0.05% trypsin DME-L Solution, nonessential amino Acids and EBSS were from Invitrogen, Paisley, UK f Fetal bovine serum gold received inactivated Warmth. was developed by the PAA, Yeovil was obtained, Gro Britain trypsin neutralizing L solution from Lonza, Wokingham, UK AL 0.4% trypan blue solution and a 1% L solution were obtained by Alcian purchased from Sigma, Poole, UK EGCG was obtained from DSM under the brand name Teavigo. EC source was in the house and was obtained by fractionation from the extract of green tea. The purity of the EC was used was 98.
4% as determined by HPLC. Maltodextrin was obtained AZD8931 from Roquette. Casein was obtained from Sigma and was used without further purification. Cell culture. HT29 and HT29 MTX E12 cells were maintained in DMEM / Glutamax containing 10% FBS and 1% NEAA. The cells were subcultured twice a week. The cells were initially by incubating First with EBSS buffer for 20 min and then with 0.05% trypsin-L Solution subcultured DTA 4 min at 37. Once the cells from tissue culture plastic were removed, trypsin DME-L was Solution in the cell suspension is neutralized by the addition of TNS. At each passage cells were seeded at 2104 cells/cm2 t. The cells were grown at 37 and 5% CO second HT29 and HT29 MTX evaluation of cell morphology and mucin production E12.
HT29 and HT29 MTX E12 cells were grown in wells of 6-well plates at 2,104 cells/cm2 seeded t. The cells were maintained in DMEM/Glutamax/10% FBS / 1% NEAA, without subculturing for different ZEITR Trees up to 21 days. sometimes sp ter ben CONFIRMS feeding the cells with fresh DMEM/Glutamax/10% FBS / NEAA 1% each day. At the time of selected Hlten points cell morphology and mucin production by both cell types were investigated. The cell morphology was examined by light microscopy using a Leica DFC320. Pictures were taken with Jasc Paint Shop Pro 7.4 software. Mucin production was measured using Alcian blue staining F: min cells were first fixed in chilled 95% ethanol / 5% glacial acetic acid for 10 min and then incubated with 1% Alcian blue / 3% acetic acid at room temperature for 5.
The cells were then washed three times with PBS to remove residual stains, and mucin production was examined by light microscopy. Paint Shop Pro 7.4 software was used to take pictures. The cell morphology was also evaluated by transmission electron microscopy. The cells were grown without a transplant, for a maximum of 21 days. On days 7, 14, 21, and the cells with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer were fixed for 90 min. After washing the cells with 1% osmium tetroxide were incubated for 30 min. The cells were then washed with 1% uranyl acetate and over night. On n Chsten morning, the samples were dried by washing with ethanol and losgel St from the underside of the plastic wells using acetone. Small pieces of the cell layer were removed and in Fl Schchen with acetone. They were then washed several times with acetone to remove residual plastic. The cells were then suspended in 50:50 TABB more hard resin / acetone and left to stir for 24 h. 50:50 resin / acetone was repl