*4.3. SiRNA Experiments*

Cells were transfected with Lipofectamine 2000 purchased from ThermoFisher. For CD82 expression knockdown, the following duplex was used: GCTGGGTCAGCTTCTA-CAAdTdT and TTGTAGAAGCTGACCCAGCdCdG. Briefly, cells were plated on 6-well dishes at 70–80% confluence the day prior to the transfection. For each well, 5 μL of Lipofectamine 2000 were added to 250 μL of Opti-MEM and the CD82 siRNA was added to a final concentration of 100 pM in 250 μL of Opti-MEM. The two samples were incubated at room temperature for 20 min and then mixed together. The complete medium of each well was then exchanged for 2 mL of Opti-MEM and the transfection mixture was added to each well. After 8 h, the Opti-MEM was removed and 2 mL of complete medium was added to each well. The following day, cells were detached and plated on 25 mm coverslips for further single molecule tracking experiments. After the experiments, the efficiency of CD82 siRNA was assessed by flow cytometry on cells used for the tracking.
