Control cells were treated with vehicle (water). In the majority of experiments, cells derived from prepared P0-cells were treated with α-amylase (P1-cells). As already mentioned, remaining P0-cells were

further cultivated after a first seeding and could be harvested a second time (second seeding). All these cells were called P1-cells. About half of the independently performed experiments selleck screening library (3 out of 7 for F344; 3 out of 6 for Lewis) were done in a blind fashion, meaning that the experimenter, who did the treatment and cell counting, was not aware about the treatment groups. In the first set of experiments, the experimenter knew about the treatment groups to be able to notice cellular alterations during α-amylase treatment. Experiments were evaluated individually and could be analyzed Temsirolimus datasheet together because no differences were observed

between blind- and non-blind-performed investigations. α-Amylase treatment in human mammary epithelial mTOR kinase assay cells The effect of α-amylase in mammary cells of human origin was studied in primary HBCEC (mammary carcinoma excisions). α-Amylase treatment was performed once per day for 2 days with 0.125 U/ml, 1.25 U/ml, 12.5 U/ml, and 125 U/ml. Control cells were treated with water. SA-β-galactosidase assay Expression of senescence-associated-β-galactosidase (SA-β-gal) is increased in senescent cells [36]. To determine if α-amylase treatment causes a change in cell senescence, primary rat mammary cells were cultured on Matrigel®-coated 24-well-plates. Treatment with salivary α-amylase (5 and 50

U/ml) for 2 days started after 1 (P1) or 4 (P2) days in culture. The cells were fixed with 1x Fixative Solution, containing 20% formaldehyde and 2% glutaraldehyde and stained against SA-β-gal for 24 h/37°C in the dark according to the manufacturers protocol and recommendations (Senescence SA-β-galactosidase Staining Kit, Cell Signaling Exoribonuclease Technology, New England Biolabs, Frankfurt, Germany). The staining was proportional to the amount of substrate (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) enzymatically transformed. Following two washes with PBS, the differentially-stained cell cultures were documented by phase contrast microscopy using Olympus imaging software cell® (Olympus, Hamburg, Germany) and quantified by counting. Cells from F344 (P1 and P2) and Lewis (only P2) were counted in three different wells and portion of SA-β-gal-positive cells was determined (one well). Positive and negative cells were counted in 6-9 sections. Data are shown as percentage SA-β-gal-positive cells. Total cell numbers per group of 759-963 cells for P1 and 510-803 cells for P2 were counted. In addition to this, cells from a human breast tumor (MaCa 700) were also treated with α-amylase (0.125, 1.25, 12.5, and 125 U/ml) and used for a SA-β-gal assay (three sections per treatment). Total cell numbers of 266-691 cells were counted.