Optimal Percoll concentration facilitates flow cytometric analysis for annexin V/propidium iodine-stained ischemic brain tissues.
We sought to determine the optimal Percoll concentration for ischemic rat brain prepared for flow cytometric (FC) measurements. Animals were subjected to the right middle cerebral artery (MCA) occlusion, and were euthanized at 3, 12, 24, and 72 h after reperfusion onset. The brains were processed by different concentrations (unisolated, 20, 25, 30, or 40%) of Percoll and stained with annexin V/propidium iodine (PI). Ischemic brain damage was evaluated by FC analysis and image analysis for histologic sections. The relative susceptibility of different phenotypes of cells to necrotic and apoptotic damage were evaluated by the FC analyses for the immunohistochemistry, PI, and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-processed brain tissues. Our results showed that FC analysis effectively detected the extent and maturation of apoptotic/necrotic brain damage, and the results were consistent with those determined from histologic brain sections. Neuron was more vulnerable to apoptosis than glia, whereas both cellular phenotypes were compatible in susceptibility for necrotic cell death. Percoll at a low concentration (20%) could effectively remove tissue debris without affecting membranous integrity of the injured neurons. Conversely, high percentages of Percoll (30-40%) substantially increased membranous damage for the injured cells. These results supported the application of FC to determine the extent and progression in time, as well as relative phenotypes of apoptotic/necrotic cell deaths following ischemic damage. We highlighted the use of Percoll at low percentages to facilitate the removal of tissue debris and to improve membrane integrity preservation for the injured neurons.
Neurophysiology Laboratory, Neurosurgical Service, Department of Surgery, National Cheng Kung University Medical College and Hospital, Tainan, Taiwan.
Wei-Sheng
WS
Juan
Hsiao-Wen
HW
Lin
Ying-Hsin
YH
Chen
Hung-Yi
HY
Chen
Yu-Chang
YC
Hung
Shih-Huang
SH
Tai
Sheng-Yang
SY
Huang
Tsung-Ying
TY
Chen
E-Jian
EJ
Lee
D000818:Animals; D017304:Annexin A5 / Q000737:chemistry; D017209:Apoptosis / Q000502:physiology; D004195:Disease Models, Animal; D005434:Flow Cytometry / Q000379:methods*; D020244:Infarction, Middle Cerebral Artery / Q000473:pathology*; D002546:Ischemic Attack, Transient / Q000473:pathology; D008297:Male; D009336:Necrosis; D009457:Neuroglia / Q000378:metabolism / Q000473:pathology*; D009474:Neurons / Q000378:metabolism / Q000473:pathology*; D011205:Povidone / Q000737:chemistry; D011419:Propidium / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D012822:Silicon Dioxide / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods
D017304:Annexin A5; D011419:Propidium; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone