Fitc anti human cd44 antibody

For generating stable MCF-7 cells expressing MMP9 (designated MCF7-MMP9), MCF-7 cells (10⁵) were seeded in the wells of a six-well plate and incubated in DMEM medium supplemented with serum for 24 h at 37°C under 5% CO₂. The cells were then transfected with a pCMV MMP9 plasmid (Sino Biological, Beijing, China) using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer's instructions. Forty-eight hours post-transfection, the cells were treated with 150 µg/ml of hygromycin (Thermo Fisher), followed by an incubation of 4 weeks at 37°C under 5% CO₂. The expression of MMP9 in MCF7-MMP9 transfected cells and MCF-7 control cells was determined using a gelatin zymography assay, as described below [52 (link)]. CD44 expression was evaluated using FITC anti-human CD44 antibody (BioLegend, CA, U.S.A.) and was detected using a BD Accuri C6 flow cytometer (BD Biosciences, CA, U.S.A.).

The quantities of secreted MMP9 were assessed using a gelatin zymography assay, as previously described [52 (link)]. Human MCF7-MMP9 or HT1080 cells (2 × 10⁴) were grown overnight on a 96-well plate at 37°C under 5% CO₂. Thereafter, the cells were treated for 24 h with 1 µM N-TIMP2_WT, C9, C9-PEX, PEX, A-C9-PEX, C9 and PEX together, or C9 and A-C9-PEX together in the relevant serum-free medium. The cell medium was collected, centrifuged for 5 min at 10,000g, and subjected to a gelatin zymography assay, as described above. To monitor the effect of different concentrations of C9-PEX on MMP9, the same process was performed using different concentrations of C9-PEX (i.e. 10 µM, 5 µM, 2 µM, 1 µM, 100 nM and 10 nM) in the relevant serum-free medium. In addition, the expression of endogenous CD44 on the HT1080 cell membrane was verified using FITC anti-human CD44 antibody (BioLegend) and was detected using a BD Accuri C6 flow cytometer (BD Biosciences).