Dq type 1 collagen

To test collagenase activity in the conditioned media, 50,000 gel-coated D1 mouse MSCs were cultured in 200 μl complete medium ±100 ng ml⁻¹ murine TNF-α for 3 days at 37 °C. Each conditioned medium (100 μl) was then mixed with 100 μg ml⁻¹ DQ-collagen type I (D12060, Thermo) that generates fluorescence upon degradation and incubated for 1 day at 37 °C ± 1 mM APMA (A9563, Sigma). Fluorescent signals were then acquired using a PHERAStar microplate reader (BMG LABTECH) with excitation/emission of 488/520 nm. To visualize collagen degradation relative to cells, gel-coated or uncoated D1 mouse MSCs were embedded in 1.25 mg ml⁻¹ collagen-I matrix containing 100 μg ml⁻¹ DQ-collagen and cultured in complete DMEM medium ± 100 ng ml⁻¹ murine TNF-α overnight. 1 mM APMA was added to each sample to completely activate latent MMPs. In some samples, the pan-MMP inhibitor GM6001 (CC1010, EMD Millipore, 10 μM) was added to test whether collagen degradation is attributed to MMPs. The fluorescent signal from DQ-collagen degradation was captured using a Zeiss LSM 770 confocal microscope under 20×/0.8 M27 Plan-Apochromat objective at excitation/emission of 495/515 nm. Z-stack images were captured along 40 μm total depth at 0.77-μm steps. Imaris was used for 3D reconstruction of z-stack images, followed by quantification of total fluorescent volumes per field of view.