Protein Protocols Protein Protocols

Flow Cytometric Protein Quantitation 49
4. In some cases methanol fixation may result in loss of the protein antigen. In this case
alternative fixation procedures should be compared. Fixation by 0.5% formaldehyde followed
by permeabilization with 0.1% Triton X-100 has also been used.
5. Fixed cells have been stored at –20°C for >1 yr with negligible loss of T Ag. However,
stability during storage must be evaluated for each antigen.
6. Monoclonal antibodies or affinity-purified polyclonal antibodies are preferred. Polyclonal
antisera may have additional antibodies of unknown specificity that will increase background
staining.
7. Antibody binding is actually a quantitative measure of the epitope recognized by the antibody.
If this epitope is masked through protein conformation/association or as an artifact
of fixation the epitope may not be detected.
8. Use of a swinging bucket microcentrifuge reduces cells loss.
9. Adjust time and temperature to maximize the signal over background. Incubation times
generally range from 1 or 2 h to overnight. Gentle rocking is recommended.
10. Primary antibodies that are directly conjugated to fluors are commercially available. In
addition, kits that allow conjugation of Alexa fluors to antibodies can be obtained from
Molecular Probes. If particularly weak signals are encountered, the signal can amplified
by additional layers of fluorescently tagged antibody. Cells stained with an FITC-labeled
primary or secondary antibody are reacted with Alexa 488-labeled rabbit anti-fluorescein
followed by Alexa 488-labeled goat anti-rabbit IgG.
11. Filtering of samples removes cell aggregates that may clog the tubing in the flow cytometer.
If samples are stored and then reanalyzed, filtering should be repeated.
12. We use a Cytofluorograph Model 50 H-H with an air-cooled argon laser (model 532,
Omnichrome, Chino, CA). The Cyclops analysis program (Cytomation, Fort Collins, CO)
was used for data analysis.
13. Use a 535-nm band pass filter for this photomultiplier.
14. Use a 640-nm long pass filter for this photomultiplier.
15. Prepare a stock of spleen cells from a healthy mouse; fix and store in 90% methanol/10%
PBS at –20°C. Prior to each run wash and stain 1 × 10 6 cells with PI only.
16. Flow cytometric standards can be developed following the procedure of Frisa et al. (7). In
brief, cell lines known to express different quantities of the protein are counted and divided
into two portions. One is fixed for flow cytometry. The other is lysed and co-run on SDS-
PAGE with known amounts purified protein in neighboring lanes. The immunoblot of the
gel and the fixed cells are stained with the same antibody. On the immunoblot, comparison
of the lysate band with the standards allows calculation of the average amount of
protein per cell. This value can then be paired with mean fluorescence of the population to
calculate a value for fluorescent units per protein molecule.
Fig. 1. (continued) forming units per cell. Cells were trypsinized and fixed at 48 h post-infection.
The sloped horizontal line in each panel was set to divide T Ag positive from T Ag negative
cells. Vertical lines indicate gates that were set on the basis of DNA content to discriminate
cells within the different phases of the cell cycle. The cell cycle distribution of the T Ag
expressing cells was: 27% G 1 phase, 9% S phase, 14% G 2/M phase, and 50% >G 2 phase. Levels
of T Ag expression in G 1 phase cells cover a 10-fold range, but only cells expressing higher
amounts of T Ag enter S phase. The cells in >G 2 phase are infected cells that are replicating
viral DNA and re-replicating cellular DNA (8). The T Ag negative G 1 phase cells serve as an
internal negative control and have the same anti-T Ag fluorescence as the uninfected G 1 phase
cells in A. (C) The relative quantities of T Ag within each cell cycle gate, as determined by
anti-T Ag staining. The bar graph shows the average quantity of T Ag per cell in the T Ag
expressing population.