Anti cd59 alexa fluor 647 mem 43

T24 wild-type and CD59-mGFP-positive cells were detached from the culture plates by treating them with accutase (Sigma-Aldrich). Cells were then washed with precooled staining buffer (HBSS containing 2% FCS) and incubated on ice for 30 min with 30 μg/mL anti-CD59-Alexa Fluor™ 647 (MEM-43) (AbD Serotec, Hercules, CA, USA), then washed twice with the staining buffer stored on ice prior to experiments. Cells were used at a concentration of 6 × 10⁵ cells/staining. Samples were analyzed on an SH800S Cell Sorter (Sony, Tokyo, Japan) and the data were further processed with the FlowJo software (Treestar, Ashland, OR, USA). Living single cells were gated according to their forward- and side-scatter characteristics.

antiCD59-546-ss488 was generated by conjugating anti-CD59 (MEM43) (Abcam, United Kingdom) simultaneously to NHS-ss-ATTO488 (ATTO-TEC, Germany) and NHS-AlexaFluor546 (Molecular Probes, Waltham, MA). Following conjugation the antibody was separated from unreacted reagents using size-exclusion chromatography. Antibodies and reagents were obtained from the following sources; AntiCD59-AlexaFluor647 (MEM43) (AbD Serotec, United Kingdom), mouse monoclonal (AP.6) against alpha adaptin used for immunofluorescence and immunoprecipitation (Abcam), mouse monoclonal against alpha adaptin used for western blots (BD, Franklin Lakes, NJ), rabbit polyclonal anti-caveolin 1 (BD), mouse monoclonal anti-flotillin 2 (BD), rabbit polyclonal against clathrin heavy chain (Abcam), Streptavidin-HRP (Pierce, Rockford, IL), PI-PLC from Bacillus cereus (Life Technologies, Waltham, MA), Streptavidin-488, -546 or -647 (Molecular Probes), Transferrin-546 or -647 (Molecular Probes), Cholera toxin subunit B (CTB) -647 (Molecular Probes), FM1-43FX (Molecular Probes), SNAP-surface 549 (NEB), BG-SS-488 and BG-SS-549 were kindly provided by our collaborators in NEB.