Gibco opti mem 1 reduced serum media

To further investigate MMP-9 activity in BMSCs, siRNA knockdown of MMP-9 was performed. Non-targeting siRNA served as control. Transfection was performed on adherent cells. Therefore, a cell number of 160,000 BMSCs was seeded in a T25 cell culture flask (Corning Inc., Corning, NY, USA) two days before transfection.
For transfection, 22.5 µL of RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) transfection reagent was diluted with 375 µL of Gibco Opti-MEM I Reduced Serum Media (Thermo Fisher Scientific). In a separate tube, 6.25 µL siRNA with a stock concentration of 20 µM (MMP-9 siRNA or non-targeting siRNA) was diluted with 375 µL Gibco Opti-MEM I Reduced Serum Media. For the formation of reaction complexes of siRNA and transfection reagent, both were mixed and incubated at RT for 5 min. To prepare the cells for transfection, the regular stem cell culture medium in the T25 cell culture flasks was replaced with 5 mL Gibco Opti-MEM I Reduced Serum Media without antibiotics. For each flask, 750 µL of transfection mix was added to the cells. Cells were used for further analyses 24 h after incubation at 37 °C in a 5% CO₂ humidified atmosphere.

HeLa, A673 and MCF10A cells were transfected with 50 nM siRNAs (Supplementary Table 6) using DharmaFECT reagent 1 and Gibco Opti-MEM I Reduced Serum Media (#31985047 Thermo Fisher). Cells were harvested 72 h after transfection and analyzed by flow cytometry, as described below. Protein and mRNA levels were assessed by immunoblotting and quantitative RT-PCR, respectively.