Analyses of cell populations in lymphoid organs by flow cytometry. (A) Strategy of antibody stain and analyses by flow cytometry is outlined by examples of histograms obtained from splenocytes to determine TCR⁺CD4⁺ cells and TCR⁺CD4⁺CD25⁺ (see also Figure 6) in DO11.10 mice. (B-I) Quantitation of TCR⁺CD4⁺ cells 60 days after gene delivery by flow cytometry. Representative examples of FACS contour plots are shown for individual samples of viable lymphocytes from lymphoid organs of DO11.10 mice. The mice received either AAV-EF1α-GFP (B,D,F,H) or AAV-EF1α-ova (C,E,G,I). Percent dual-positive cells (top right quadrant) are indicated. Note that AAV-EF1α-ova–transduced mice showed significant reduction in TCR⁺CD4⁺ cells compared with control animals. Antibody stain was PE-Cy5.5–conjugated anti-CD4 and FITC-conjugated KJ1-26. Portal nodes had been pooled from 4 mice prior to flow cytometry.