LSC Application: Cancer Research / Gene Therapy: Adenoviral infection and p53 protein expression

The assessment of adenoviral infection and p53 protein expression is useful for the analysis of adenoviral vectors containing and expressing the wild-type protein encoded by the p53 gene.

Indicator cells such as the human embryonic kidney line 293 are experimentally infected with the adenoviral vector for a single viral replication cycle. Infected cells are fixed and stained for intracellular protein analysis using specific antibodies labeled with fluorochromes. Analysis can be performed either in univariate mode, i.e., a single antibody for either adenoviral infection or p53 protein, or in bivariate mode for both signals simultaneously. Bivariate LSC analysis allows the investigator to correlate within each cell scanned whether the cell has been infected by the adenoviral vector, and the p53 protein expression status of the cell. The use of area analysis combined with laser light scattering can be used to discriminate multiplets in order to reduce non-true two-color artifact. CompuSort™ relocation is used for verification of true two-color cells and image capture.

adenovirus infected human cells Determination of single-cell events. Adenovirus-infected human 293 cells were analyzed using 488 nm laser light scatter analysis on the LSC. Events falling within the gate shown were determined empirically to be single cells.

microscopy of p53 infected cells laser scanned p53 infected cells

LSC analysis of human 293 cells infected with rAd5-CMV-p53. Two galleries (B & W) of intracellular positive p53 cells relocated by CompuSort. The first gallery is the light microscopy of the cell, and the second is the orange fluorescence of the same cell. This demonstrates that the positive events within the gate are truly orange positive.

hexon protein and p53 dot plot
Bivariate analysis of intracellular hexon protein and p53 protein in adenoviral infected human 293 cells. cytoplasmic and nuclear fluorescence staining
True two-color cell (in the center) relocated from the upper right-hand population in the bivariate FITC vs. PE scattergram. This picture was taken through a blue filter so that green fluorescence is green. Red bleed-through appears as yellow-orange. The intracellular p53 is mainly nuclear associated as expected; the intracellular hexon staining is cytoplasmic as expected. Three other cells within the field were also relocated as true two-color, and three cells as single (green) color.

kinetics of p53 expression A multiplet cell from just outside of the Area vs. LSMP gate showing how two-color artifacts can occur. These are actually both two-color cells, but one is red-hi and the other green-hi. Two cells are infected but at two distinct times in the kinetics for p53 protein expression, i.e., very early and much later. The green overpowers the red in this doublet. Further investigation of this event by CompuSort showed that if one were looking at this as a single event, such as a flow cytometer would have done, it would appear to be green-hi and red-dim. Thus, it would have been questionable, or a negative call, for a "real" two-color event. This demonstrates the power of the area discrimination field and the use of CompuSort to investigate bivariate staining to more accurately quantitate and help eliminate potential artifacts.

Sample preparation
Human embryonic kidney cell line (293) infected cells are harvested by brief trypsinization and fixed using cold methanol/acetone (1:1). Cells are washed, pelleted, and stained using 1.5 µg of FITC-anti Ad5 hexon mAb (Chemicon) diluted in PBS (37° C for 50 minutes). The cells are then washed, pelleted, and stained using 0.5 µg of PE-anti human p53 mAb (DO-7, PharMingen) diluted in PBS (23° C for 30 minutes). The cells are then washed, pelleted, and resuspended in a 50% Fluoromount G/PBS suspension (Southern Biotechnology Associates). An aliquot, usually 10 µL, is applied to a clean glass slide and the sample is coverslipped for analysis. Sample is kept at room temperature in the dark until reading.

LSC Analysis
The prepared slide is placed on the LSC stage and the entire cover slip area is selected for scanning analysis. Slides are scanned at 20X objective using the argon laser line. Laser light-scatter events are captured and used to contour single cells within the scan data display. Contour discrimination was set from the nuclear portion of the scanned cell such that about 67% of the total light scatter pixelation was contoured. Green (FITC) and orange (PE) PMT-detector gain voltages are set so that no greater than 75% maximum saturation of the max pixel in the respective fields is achieved during the scan. An area vs. laser light scatter max pixel scattergram is collected on the scan and CompuSort used to empirically set an area discrimination gate so that multiplets are excluded from analysis.

Data Interpretation
Green fluorescence is associated with adenoviral infection (hexon protein), and orange fluorescence is associated with increased p53 protein expression. Hexon protein is not expressed in uninfected cells; however, p53 protein (background) will be present in most cell lines. Data can be analyzed in either integrated log fluorescence mode or in linear max pixel mode.

Procedure and data from Dr. Michael Grace, Schering-Plough Research Inst., Kenilworth, N.J. Ref. Cytometry. 1998 Nov; 33: 290-296.