5 laser lsr 2

Three million cells per sample were added to BD Falcon 5 mL polypropylene round-bottom tubes. Cells were washed with 2 mL of 1X PBS and centrifuged at 1200 rpm for 5 minutes at 4°C. Supernatant was removed and cells were washed in 2 mL of FACS buffer (1X PBS + 3% FBS). Supernatant was removed and 3 μL of Fc Receptor Block (BD) was diluted in 50 μL of FACS buffer per sample and incubated for 15 minutes at 4°C. Cells were washed with 2 mL of FACS buffer and centrifuged at 1200 rpm for 5 minutes at 4°C. Supernatant was removed and antibodies were diluted in 50 μL of FACS buffer and incubated for 30 minutes at 4°C. Cells were washed with 2 mL of FACS buffer and centrifuged at 1200 rpm for 5 minutes at 4°C. If biotinylated antibody was used, streptavidin was diluted in 100 μL FACS buffer per sample and incubated for 30 minutes at 4°C. Viability staining was performed by adding Tonbo Ghost Dye UV450 (1:500) or DAPI (1:1000). Cells were analyzed on the 5-Laser BD LSR II or sorted on the 5-Laser FACS Aria III in the VUMC Flow Cytometry Shared Resources.

After methanol permeabilization, cells were stained with 1:250 anti p-ACC antibody (Cell Signaling) for 30 minutes in the dark at room temperature. Cells were then stained with 1:1000 Donkey anti-Rabbit Ax647 (Life Technologies) for 30 minutes in the dark at room temperature, and were washed and resuspended in PBS for analysis on 5-laser BD LSRII (BD Biosciences, San Jose, CA) at the Vanderbilt Flow Cytometry Shared Resource and evaluated using Cytobank software. Untreated cells were stained with only the secondary antibody and used as negative control.