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

Korean Journal of Pediatrics 2005;48(5):545-550.
Published online May 15, 2005.
Single Cell Dissociation Methods for Flow Cytometric Cell Death Analysis of Hypoxia-Ischemia Injured Newborn Rat Pup Brain
Jong Hee Hwang1, Dong Kyung Sung2, Chang Won Choi1, Saem Kang2, Yun Sil Chang1, Won Soon Park1, Munhyang Lee1
1Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
2Samsung Biomedical Research Institute, Seoul, Korea
저산소성 허혈성 뇌손상이 유발된 신생백서에서 단일세포의 분리
황종희1, 성동경2, 최창원1, 강샘2, 장윤실1, 박원순1, 이문향1
1성균관대학교 의과대학 삼성서울병원 소아과
Won Soon Park, Email: wspark@smc.samsung.co.kr
: 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.
: 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).
: 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.
: 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.
Key Words: Cell separation , Flow cytometry , Hypoxia-ischemia , Brain , Animals , Newborn

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