Figure 6. The effect of lipofuscin autofluorescence on human brain DNA content histograms.

Lipofuscin is a broad-spectrum autofluorescent pigment seen in both rhodamine and fluorescein microscope channels that accumulates at the nuclear periphery in some aging neurons and might contribute autofluorescent signals in DNA content flow cytometry experiments (Riga and Riga, 1995). This possibility was assessed on nuclei with low lipofuscin (a–d, Lipo-) vs. high lipofuscin (e–h, Lipo+) pigment levels, stained with PI (red); Lipo+ nuclei were visible in a fluorescein channel (green). Autofluorescence from lipofuscin can be minimized with the addition of the lipophilic dye, Sudan Black (i–l, SB), which preferentially binds to lipofuscin granules (black particles in k and l) (Schnell et al., 1999) and quenches the autofluorescent signal, and then is visualized by brightfield microscopy. SB staining was used in initial experiments; however further analyses demonstrated that it was not necessary (m–o), wherein the addition of SB to human brain nuclei prior to FCM did not significantly alter DNA content histograms (SB- (red histogram), SB+ (black histogram), and overlay). Importantly, when unstained isolated nuclei were analyzed by FCM, no signal from lipofucsin alone (p, unstained) was observed in the PI (or DRAQ5, data not shown) channel indicating that the presence of lipofuscin does not contribute to fluorescence signals in DNA content FCM of human brain nuclei. Scale bar = 5 μm in a–l.