Flow cytometric analysis of electronic nuclear volume and DNA content in normal mouse tissues.
Light scatter is used in flow cytometry for identification of cells based on their size and/or granularity. However, forward light scatter is not an accurate measure of cell size. The measurement of Electronic Volume (EV) by Coulter principle is more accurate. However, EV cannot be measured on most of the commercially available flow cytometers. We have described the development and applications of a flow cytometer that can simultaneously measure Electronic Nuclear Volume (ENV) and DNA content. In the present study we have used a commercially available NPE Quanta for measuring EV and DNA content of different normal mice tissues. Fresh/frozen or formalin fixed-paraffin embedded tissues from mice were processed for isolation of nuclei, which were then analyzed for EV versus DNA content. By using these two parameters, distinct sub-populations were identified in liver, thymus, small intestine and bone marrow. Dual parametric analysis of EV versus DNA content can be a valuable technique for identification of sub-populations in heterogeneous cell mixtures such as those of complex tissues like bone marrow, intestine and tumors. The methods established are rapid and can provide valuable data for identification and characterization of sub-populations for cell cycle analysis by flow cytometry.
Krishan
Awtar
A
Division of Experimental Therapeutics, Radiation Oncology Department, University of Miami, School of Medicine, Miami, Florida 33101, USA. akrishan@med.miami.edu
Cabana
Raquel
R
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002453:Cell Cycle; D002467:Cell Nucleus; D048429:Cell Size; D004247:DNA / Q000032:analysis*; D004583:Electrons; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005557:Formaldehyde; D005629:Frozen Sections; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010179:Pancreas / Q000166:cytology / Q000378:metabolism; D016612:Paraffin Embedding; D013950:Thymus Gland / Q000166:cytology / Q000378:metabolism
D005557:Formaldehyde; D004247:DNA