Zinc fixation preserves flow cytometry scatter and fluorescence parameters and allows simultaneous analysis of DNA content and synthesis, and intracellular and surface epitopes.
Zinc salt-based fixation (ZBF) has proved advantageous in histochemical analyses conducted on intact tissues but has not been exploited in flow cytometry procedures that focus on quantitative analysis of individual cells. Here, we show that ZBF performs equally well to paraformaldehyde in the preservation of surface epitope labeling and forward and side scatter parameters as measured by flow cytometry. ZBF-fixed mouse epithelial keratinocytes exhibit a staining pattern for the surface markers Sca-1, CD34 and alpha6 integrin that is highly analogous to live cells. Furthermore, ZBF also preserves DNA allowing subsequent quantitative PCR analysis or labeling for incorporation of the thymidine analog EdU following surface and intracellular epitope staining. Finally, ZBF treatment allows for long-term storage of labeled cells with little change in these parameters. Thus, we present a protocol for zinc salt fixation of cells that allows for the simultaneous analysis of DNA and intracellular and cell surface proteins by flow cytometry.
Jensen
Uffe Birk
UB
Department of Clinical Genetics, Aarhus University Hospital, 8000 Aarhus C, Denmark. uffe@humgen.au.dk
Owens
David M
DM
Pedersen
Søren
S
Christensen
Rikke
R
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D004247:DNA / Q000032:analysis*; D000939:Epitopes / Q000032:analysis*; D005404:Fixatives / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D042541:Intracellular Space / Q000378:metabolism*; D051379:Mice; D012313:RNA / Q000032:analysis; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods*; D015032:Zinc / Q000378:metabolism*
D015415:Biomarkers; D000939:Epitopes; D005404:Fixatives; D012313:RNA; D004247:DNA; D015032:Zinc