Signaling Protocols

• Fixation of BMDM for Phospho-FACS Analysis
• Staining of Fixed BMDM

Fixation of BMDM for Phospho-FACS Analysis

1. Plate BMDM in wells in a 6-well plate at density of 1 X 10⁶ cells/well.
2. Perform experiment (Add ligands, bacteria etc) and save some supernatant for subsequent analysis by ELISA if desired.
3. Remove supernatant from cells by aspiration.
4. Fix cells by adding 1 mL of PBS + 1.6 % paraformaldehyde and incubate at room temperature for 10 minutes.
5. Aspirate fix and wash cells by adding 2 mL PBS and remove by aspiration.
6. Add 1 mL PBS + 1 mM EDTA (PBS/EDTA) and place plates on ice.
7. Scrape fixed cells up into the PBS/EDTA using a cell scraper.
8. Remove cells suspended in 1 mL PBS/EDTA to a microcentrifuge tube.
9. Freeze at -80^°C until ready to stain.

Staining of Fixed BMDM

Cells were thawed, then spun down and resuspended in cold MeOH, and kept at 4 degrees for 10 minutes. Cells were then barcoded, with each time point for a given stimulation stained with a unique dye pattern, so that all the time points could be pooled. The 0 minute time points were stained with 0.33 PacBlu succinimidyl ester (Invitrogen), 5 minutes with 2 ug/ml PacBlu, 10 minutes with 0.33 ug/ml PacOrange, 20 minutes with 0.33 ug/ml PacOrange and 2 ug/ml PacBlu, 40 minutes with 2 ug/ml PacOrange, and 60 minutes with 2 ug/ml PacOrange and 0.33 ug/ml PacBlu. Staining with succinimidyl esters was done in a PBS to MeOH ratio of 2:1 at room temp for 20 minutes. Cells were washed and pooled, then stained with a phospho-specific antibody. Cells were than analyzed with an LSR2 flow cytometer.