Generating fluorescently labeled reagents

C9 (CompTech) was chemically labeled with AlexaFluor-568 labeling kit (Thermo Fisher Scientific) as per manufactures instructions. Briefly, 250 μL of 1 mg/mL C9 was mixed with 50 μL of 1 M sodium bicarbonate. The mixture was then added to 1 vial of Alexafluor-568 reagent and stirred at room temperature for 1 h. The labeled protein was purified from the free label by spinning at 1000 x g for 2 minutes through a Zeba Dye removal column (Thermo Fisher Scientific). The resin was then washed with 2 × 250 μL of phosphate buffered saline (PBS) to recover the C9 conjugate. Fractions were combined, aliquoted, flash frozen and stored at −80 °C for future use.

To generate cells expressing fluorescently labeled CD59, we transfected SNAP-tagged CD59 into Chinese hamster ovary-K1 (CHO-K1) cells (a gift from A. Kusumi). Cells were cultured in Hams-F12 media (Thermo Fisher Scientific) with 10% fetal bovine serum (FBS). Approximately 250,000 cells were seeded into 6 well plates and allowed to adhere overnight. Cells were transfected with 4 mg of SNAP-CD59 (Addgene) using Lipofectamine 3000 (Life Technologies). After 24 hours cells were detached with 1 mL of PBS supplemented with 1 mM EDTA. Cells were replated on poly-l-lysine coated 8-well chamber slides at 50,000 cells per well and allowed to adhere overnight. To stain for CD59, a 1 in 200 dilution of SNAP-Oregon cell permeable ligand (NEB) was added.