Heat-Assisted Flow Cytometry

The limitations of the application of conventional detergents mentioned above can be circumvented by replacing this approach with cell membrane permeabilization by microwave heating. Improved detection of intracellular antigens can be obtained with microwave heating used in combination with flow cytometry. This approach yields histogram patterns that show clear discrimination between intact cells and cell debris (Fig. 9.5). 

Accurate flow cytometric measurements depend on clear discrimination between intact cells and cellular debris. Both cytoplasmic (bcl-2, CD68, lipocortin-1, a-smooth muscle actin, and desmin) and nuclear antigens (p53, PCNA, Ki-67) have been simultaneously detected with this combined protocol (Lan et al., 1996 Millard et al., 1998). 

The cells are incubated in the primary antibody at an appropriate dilution for 30-60min at 4°C or room temperature, depending on the type of cells or antigens. After being washed in PBS, the cells are incubated in a fluorescein isothiocyanate (FTTQ-con-jugated goat antimouse IgG (or sheep antimouse IgG) for 30 min at 4°C in the dark. The cells are washed twice in PBS and resuspended in —200 (xl of 1% fetal calf serum or ISOTON II for flow cytometric analysis. 

The nuclear isolation method has been used for processing archival paraffin-embedded mammary tumors for monitoring estrogen expression and aneuploidy (Sabe et al., 1999). These two parameters have important diagnostic and prognostic significance in mammary tumors. 

The cells and nuclei (0.5-1.0 X 106) are aliquoted into polystyrene tubes. Approximately 10 jjlI of normal horse serum (Vector Laboratories, Burlingame, CA) is added to block any nonspecific binding. The suspension is incubated with biotinylated antiestrogen monoclonal antibody (1D5, Dako Corp., Carpinteria, CA) at 1 25 dilution for 1 hr at 37°C. Aliquots are stained with 10 p,l of fluorescein isothiocyanate (FITC)-conjugated strepta-vidin (Dako buffer containing 0.1% Triton X-100). 

---