Growth factor reduced phenol free matrigel

A matrigel/collagen mixture was prepared using growth factor reduced, phenol-free matrigel (Corning) and DQ^™ collagen, type I, fluorescein conjugate (Life Technologies) at a 10:1 ratio, maintained on ice. 12-well glass bottom plates (Mat Tek) were coated with 80 μL of the basement membrane mixture, and were allowed to equilibrate at 37°C in humidified conditions with 5% CO₂ for 1 hour to solidify. 1 × 10³ cells per group were suspended in 350μL of basement membrane solution and added per well. 3D cultures were allowed to equilibrate for 2 hours to solidify prior to adding media containing reduced serum (0.5%) and either DMSO control, MMP8 inhibitor I (10 μM, EMD Millipore), or IKK2 inhibitor IV (10 uM, EMD Millipore). After 48 hours, cells were carefully washed with PBS 3×, fixed in 4% paraformaldehyde for 30 minutes, washed 3× with PBS, and stained with Texas Red^®-X Phalloidin and DAPI (Life Technologies) (fixation and staining performed at room temperature). Cells were imaged using a laser-scanning confocal microscope (LSM 510 META; Carl Zeiss). Dapi and Phalloidin staining were used to locate B cell lymphoma colonies. Enzyme-driven hydrolysis of collagen was characterized by positive fluorescent signal derived from fluorescein per individual cell/colony. Positive or negative fluorescein expression was manually quantitated.