Protein Protocols Protein Protocols

Flow Cytometric Protein Quantitation 47
antibody. If similar cells not expressing the protein of interest are available they can be
stained at the same dilutions to determine levels of nonspecific staining.
3.3. Staining
1. Transfer 1.5 × 106 cells to a 1.5-mL microcentrifuge tube and centrifuge at 2000g for 15 s
(see Note 8).
2. Discard supernatant and resuspend the cell pellet in 1 mL of cold PBS. Repeat centrifugation
step.
3. Discard PBS and add 0.5-mL of diluted primary antibody. Mix gently.
4. Incubate at 4°C with gentle agitation (see Note 9).
5. Pellet at 4000g for 15 s and discard supernatant.
6. Resuspend pellet in 0.5 mL of WS and repeat step 5.
7. Resuspend pellet in 0.5-mL of diluted fluorescently labeled secondary antibody (see
Note 10). Mix gently.
8. Incubate at 37°C for 2 h with gentle mixing; minimize exposure to light.
9. Add 0.5 mL of RNase and mix gently.
10. Incubate at 37°C for 30 min.
11. Add 0.5 mL of PI, bringing the total vol to 1 mL. Mix gently.
12. Filter each sample through a 53-µm mesh nylon grid (Nitex HC3-53, Tetko, Elmsford,
NY) (see Note 11)
3.4. Flow Cytometry and Analysis
Most commercially available flow cytometers are capable of multiparameter analysis
(see Note 12). Using 20 mW of power tuned to 488 nm, minimize the coefficient of
variation (CV) for the green photomultiplier (see Note 13) to