QUANTIFYING APOPTOSIS IN BANKED HUMAN BRAINS USING FLOW-CYTOMETRY

Fragmentation of genomic DNA, a major biochemical feature of programme d cell death (apoptosis), is easily detected when apoptosis is prevale nt. In brain tissue apoptotic cells are usually scarce and detection r equires more sensitive techniques. We describe a highly sensitive meth od to quantify apoptosis in frozen human brain tissue using Row cytome try. Nuclei from homogenized brain specimens were isolated to purity u sing discontinuous isopyknic centrifugation through 2.2 M sucrose. DNA strand breaks in apoptotic nuclei were conjugated with biotinylated-d UTP using terminal deoxynucleotidyl transferase (TdT) and lagged with streptavidin-conjugated FITC (TUNEL). Negative controls excluding the TdT step, and positive controls using DNAase pretreatment to create 3' -OH strand breaks were run in parallel. The proportion of nuclei with TdT-dependent labeling in adult brain specimens was (0.01% in 6 out of 7 specimens. In 3 fetal brains it averaged 0.86 +/- 0.11%. Apoptotic cells were readily detected in 2 malignant glial neoplasms and in a pa tient with HIV encephalitis. Comparable frequencies of stained nuclei were present in adjacent specimens embedded in paraffin and labeled in situ. By screening millions oi nuclei cytometry detected very rare ap optotic events, producing quantitative results using banked frozen bra ins. The method has potential applications to studies of human brain d evelopment, neurodegenerative diseases, and brain tumors.