Out of the blue: a comparison of Hoechst side population (SP) analysis of murine bone marrow using 325, 363 and 407 nm excitation sources.
BACKGROUND
The discrimination of side population (SP) cells is a useful flow cytometric tool for the study of putative stem cells in many tissue types. Previous studies on murine bone marrow (BM) SP cells and concurrent surface immunophenotyping provide a good model for comparing Hoechst bivariate profiles. Here we compare the results of SP cells from the same specimens generated on three different instruments.


METHODS
Concurrent SP analysis and immunophenotyping was performed on murine BM. The data acquisition was performed on a DakoCytomation MoFlo, a Becton Dickinson LSR and a Becton Dickinson FACSAria to compare the ability of each instrument to discriminate putative stem cells. Further experiments examined the effect of apoptotic changes, assessed by Annexin V binding, and the presence of nucleated erythroid precursors on the Hoechst profile.


RESULTS
Identifiable and equivalent side populations were generated on all three instruments despite differences in appearance of the Hoechst profiles. Differences in laser type, collection filter combinations and the presence of erythroid precursors and apoptotic cells all contributed to this effect.


CONCLUSIONS
In our hands, murine SP cells with the expected surface immunophenotype can be identified on a MoFlo, LSR and a factory standard FACSAria.
Simpson
Catherine
C
FACS Laboratory, London Research Institute, Cancer Research UK, London.
Pearce
Daniel J
DJ
Bonnet
Dominique
D
Davies
Derek
D
D000818:Animals; D017304:Annexin A5 / Q000737:chemistry; D001562:Benzimidazoles / Q000737:chemistry*; D001854:Bone Marrow Cells / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000276:immunology*; D016130:Immunophenotyping / Q000379:methods; D007834:Lasers; D051379:Mice; D008810:Mice, Inbred C57BL
D017304:Annexin A5; D001562:Benzimidazoles; C017807:bisbenzimide ethoxide trihydrochloride