Preparation of a collagen master mix for 3D collagen I gels

To prepare a cell-free collagen I master mix of a defined volume (90 μl), 10 μl of a 10× concentrated minimal essential Eagle’s medium (MEM) (M0275, Sigma-Aldrich) was added to 5 μl of a 7.5% sodium bicarbonate (BIC) solution (25080-060, Gibco) in a sterile tube and gently mixed until the indicator color changed from yellow (acidic) to purple (basic). Subsequently, 75 μl of purified bovine collagen I stock solution (3.0 mg/ml; PureCol) (5005-C, Advanced BioMatrix Inc.) was added to the MEM/BIC solution and gently mixed until a homogeneous, slightly purple color was obtained. The collagen I master mix was kept on ice for immediate use.