Chemi doc xrs imaging device

Assays were performed to evaluate proliferation and survival. In total, 286 LS174T and 180 MCF7 cells were seeded into 24-well cell culture plates with 3% FBS, in the presence or absence of TSP1; note that TSP1 was added on floating cells, the first day. Cells were then incubated at 37 °C in a 5% CO₂ atmosphere for 7 days, then washed twice with PBS, and stained with 0.4% crystal violet. The colonies were then washed twice with water, visualized with a Bio-Rad Chemi Doc XRS Imaging device, and counted using Quantity One imaging software (Bio-Rad).

Assays were performed in control or treated cells as well as in PLCs to evaluate proliferation and survival. 500 to 1500 cells were seeded into 6-well cell culture plates with 10% FBS and incubated at 37°C in a 5% CO2 atmosphere for 7 to 10 days, then washed twice with PBS, and stained with 0.4% crystal violet. The colonies were then washed twice with water, visualized with a Bio-Rad Chemi Doc XRS Imaging device and counted using Quantity One imaging software (Bio-Rad).

200.000 cells were cultivated in a 12-well format and treated with IC50 concentrations for single or combinational treatment for 48 hours. Proteins were isolated using RIPA lysis buffer supplemented with cOmplete Protease Inhibitor Cocktail Tablets (Roche Diagnostics, Mannheim, Germany) and PhosSTOP Phosphatase Inhibitor Cocktail Tablets (Roche Diagnostics). Protein concentration was measured using Bradford reagent (Sigma-Aldrich, St. Louis, Missouri, USA). 30 µg of protein was loaded and separated on a 10% polyacrylamid gel in a Tris-Glycin-SDS buffer. Proteins were then blotted onto nitrocellulose membranes using Turboblot (Biorad, Hercules, California, USA). Membranes were blocked in 5% bovine serum albumin (Sigma-Aldrich) diluted in TBS-T and incubated with primary antibodies overnight at 4° C. Primary antibodies used were P-AKT Ser473 (Cell Signaling, #4060), T-AKT (Cell Signaling, #), P-ERK1/2 Tyr204 (Santa Cruz, sc-101761), P-ERK1/2 Thr202/Tyr204 (Cell Signaling, #9101), T-ERK 1/2 (Santa Cruz, sc-93/sc-154). Visualization was performed by using horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Laboratories, Dallas, Texas, USA) and the enhanced chemiluminescence detection system (Biorad) on a ChemiDoc XRS+ Imaging device (Biorad).