Single Cell Dissociation Methods for Flow Cytometric Cell Death Analysis of Hypoxia-Ischemia Injured Newborn Rat Pup Brain

Hwang, Jong Hee; Sung, Dong Kyung; Choi, Chang Won; Kang, Saem; Chang, Yun Sil; Park, Won Soon; Lee, Munhyang

Abstract

PURPOSE
Newborn brain tissue has to be dissociated into a single cell suspension for flow cytometric analysis of cell death during hypoxia-ischemia. Thus the development of a method to dissociate cells from the brain tissue with least damage and maintenance of membrane and antigen integrity remains the challenge for the in vivo application of this technique. We evaluated the efficacy of mechanical or enzymatic (collagenase or tryspin) methods of brain tissue disaggregation.
METHODS
The extent of the damage to the plasma membrane and loss of the characteristics of the membrane induced with each dissociation method was determined by comparing the flow cytometric results labeled with both fluorescent annexin V and propidium iodide of the newborn rat pup brain tissue in the control group (n=10) and in the 48-hour after hypoxia-ischemia group (n=10).
RESULTS
In the control group, the cell percentage of damaged, apoptotic and necrotic cells of both hemispheres with the mechanical dissociation method was significantly increased compared to the trypsin or collagenase method. In the 48-hour after hypoxia-ischemia group, the cell percentage of apoptotic and necrotic cells of the right hemisphere with the collagenase method significantly increased, and live cells significantly decreased compared to the left hemisphere, control group. Although the same trend was observed, the extent of alterations made with the trypsin method was significantly less compared to the collagenase method.
CONCLUSION
The dissociation of neonatal brain tissue for flow cytometric analysis with collagenase was most efficacious with the least cell damage and preservation of the plasma membrane characteristics.