Cellular uptake study

1.     Seed 1 x 10⁵ MDA-MB-231 cells in a 24-well plate and incubate at 37 °C for 12 hours.

2.     Wash the cells with PBS for 1 time

3.     Dilute Cy5-labeled siRNA or nanocomplex in Opti-MEM medium to a final concentration of 50 nM siRNA.

4.     Add the diluted solutions to each well and incubate the plate at 37 °C

5.     After 2, 4, and 6 hours transfection, remove the medium and wash the cells with PBS for 1 time.

6.     Dilute heparin in Opti-MEM medium to a final concentration of 100 nM, add the solution into each well, and incubate the plate at 37 °C for 20 min.

7.     Remove the medium carefully and wash the cells 3 times with 500 µL PBS.

8.     Detach the cells with trypsin and mixed with DMEM medium containing 10% FBS.

9.     Centrifuge the solution at 300g for 10 min. Remove the supernatant and suspend the cells in PBS.

10.  Transfer the cells to a 1.5 mL tube, centrifuge at 300g for 10 minutes, and resuspend the cells in PBS.

11.  Repeat step 10 for one more time. Resuspend the cells in 400 µL PBS for flow cytometry analysis.

Category