- Prepare single cell suspension.
a) Harvest bone marrow cells by flushing femur with ice-cold bone marrow harvesting medium.
b) Filter the suspended cells through 70µm cell strainer while keeping cells on ice.
c) Count the cell concentration.
d) Centrifuge the filtered cells at 1500rpm for 5-10 min.
e) Resuspend the cells with HBSS+ staining buffer (HBSS+, ~2×10^7 cells/mL if possible).
- Load FDG into cells.
a) Aliquot 100µl suspended cells for staining. Prepare controls including unstained control, single color controls for FACS compensation.
b) Prepare 2mM FDG working solution by diluting 20mM FDG stock solution with ice-cold distilled water. Prepare working FDG control solution by diluting FDG control stock solution by 1:10. Aliquote 100µL FDG working solution or control solution to amber tubes.
c) Prepare 15mL tubes with 2mL HBSS+ buffer and keep on ice.
d) Make sure all the sets of cell sample tubes, FDG tubes and 2mL HBSS+ tubes are ready for the following steps.
e) Prewarm cell sample tubes and corresponding FDG (or control) tubes in 37ºC water bath for 10min. 2mL HBSS+ tubes are kept on ice during loading.
f) Follow the FDG loading strategy shown in Figure 1 to load FDG into cells. Transfer prewarmed cells into corresponding FDG tubes. Mix thoroughly. Return to 37ºC bath for exactly 1 min. Stop the FDG loading at the end of one min by transferring mixture into 2mL ice-cold HBSS+.
g) Keep on ice for 1.5 hours to allow accumulation of FITC release from FDG in LacZ+ cells.
h) Centrifuge at 1500rpm for 7 minutes, discard supernatant and resuspend cell pellets with 100µL ice-cold HBSS+ buffer.
- Stain cells with antibodies.
a) Add 2µl antibodies to cells and slowly rock at 4ºC for 15 min.
b) Centrifuge at 1500rpm for 5-10 min, discard supernatants and resuspend cell pellets with 200ul ice-cold HBSS+.
c) Add 7µl 7-AAD solution to 200µl cells
- Perform FACS analysis.
a) Adjust voltage.
b) Run compensation.
c) Run samples and collect FACS data.
d) Analyze your FACS data.