Rapid assay for quantitative measurement of apoptosis in cultured cells and brain tissue.
Analysis of apoptosis in brain tissue following ischemia, hypoxia, or oxidative stress has technical limitations. The use of counting cells displaying apoptotic morphology is time intensive, vulnerable to sampling errors, and suffers from low numbers of total recorded events. Other cell death assays such as agarose gel analysis of DNA fragmentation, TUNEL, or ELISA are time intensive, limited to a single endpoint measure, and can be technically difficult to perform or reproduce. To overcome these limitations, we set out to develop a technique using flow cytometry to measure apoptosis based on the physical properties of light scatter produced from isolated nuclei. This dye/marker free approach would bypass many of the inherent encumbrances and reproducibility problems found in other apoptosis assays. Here we demonstrate that this new technique, using flow cytometry performed on isolated nuclei, allows rapid quantitation of apoptosis in a variety of brain tissues without the need for intercalating dyes or fluorescent markers. We conclude that this technique significantly improves currently available protocols to quantify apoptosis from tissue and offers the possibility to perform additional analysis on the same population of nuclei via downstream assays.
Department of Neurosciences and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555-1043, USA.
Bryce Patrick
BP
Portier
Diana Carolina
DC
Ferrari
Giulio
G
Taglialatela
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D001921:Brain / Q000166:cytology* / Q000502:physiology; D002467:Cell Nucleus / Q000502:physiology / Q000648:ultrastructure*; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods; D015925:Cryopreservation; D004247:DNA / Q000032:analysis / Q000378:metabolism; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D020925:Hypoxia-Ischemia, Brain / Q000175:diagnosis / Q000503:physiopathology; D008297:Male; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000502:physiology; D009488:Neurosciences / Q000295:instrumentation / Q000379:methods; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism; D016716:PC12 Cells; D011419:Propidium; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D013997:Time Factors
D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011419:Propidium; D004247:DNA