Memerald cd9 10

For transient transfection of cells for live cell imaging, we used a method that was similar to the one described above with some modifications. Briefly, cells were plated at a density of 3 × 10⁵ to 4 × 10⁵ cells per six-well plate so that cells reached 90% confluence the next day. For transfections, Lipofectamine 2000 (11668500, Thermo Fisher Scientific) was complexed with 1 μg of the plasmid mEmerald-CD9-10 (54029, Addgene), as described above. At 48 hours after transfection, mEmerald-CD9 expression was verified by using fluorescent microscopy, and cells were used for subsequent live cell imaging experiments.

The CD9_GFP fusion protein was cloned as previously described²³ (link) from mEmerald-CD9-10 (Addgene plasmid no. 54029, a gift from Michael Davidson) into the pENTR1a no CCDB. Gateway cloning with LR reaction was performed according to manufacturer’s condition using pLENTI6.3/TO/V5-Dest as destination vector (Thermo Fisher Scientific, Waltham, MA, USA). CD9_GFP pseudotyped lentiviruses were produced in HEK293FT cells. These were seeded in two T75 flasks at 8.25 × 10⁵ cells/mL and directly transfected with pLenti-CD9_GFP for gene of interest (GOI) delivery, psPAX2 (Addgene plasmid no. 12260, a gift from Didier Trono) for viral capsid proteins, and pCMV-VSV-G (Addgene plasmid no. 8454, a gift from Bob Weinberg)³² (link) for viral envelope proteins. 16 hr after transfection, sodium-butyrate-containing media (0.01 M) was added for 8 hr. Afterward, media without sodium butyrate was used and collected every 24 hr. After 5 days, media was centrifuged 30 min at 2,000 × g, filtered (450 μm), and stored at −80°C.