Centriprep ym 3 centrifugal units

Tissue-culture dishes (150 mm) were used to SMMSCs culture at 37 °C with 5% CO₂ and an atmosphere of 95% air. As the cell culture in DEMD became confluent, PBS was applied to rinse SMMSCs and the growth medium was substituted with DMEM. After 24 h, the conditioned medium was collected and centrifuged twice, first at 500×g for 10 min and afterward at 3000×g for 20 min, to remove cell debris. Finally, pre-cleaned secretome was concentrated to ~ 1.5 mL using Centriprep YM-3 centrifugal units (Millipore, Bedford, MA, USA) [30 (link)].

NIH-3T3 J2 fibroblasts (ATCC) were cultured in a 150-mm tissue-culture dish at 37°C in a humidified atmosphere of 90% air/10% CO₂. The culture was maintained in DMEM supplemented with 10% bovine serum (BCS, Gibco). When fibroblast culture became confluent, cells were rinsed with PBS and the growth medium was replaced with DMEM supplemented with 10% depleted BCS. The culture was then maintained for 24 h to condition the medium. After 24 h, the medium was collected and centrifuged at 500 x g for 10 min then at 3,000 x g for 20 min to remove cell debris. The pre-cleaned medium was then concentrated from ~120 ml initial volume to ~1.5 ml final volume using Centriprep YM-3 centrifugal units (Millipore).