Zinc fixation preserves flow cytometry scatter and fluorescence parameters and allows simultaneous analysis of DNA content and synthesis, and intracellular and surface epitopes

TY - JOUR

T1 - Zinc fixation preserves flow cytometry scatter and fluorescence parameters and allows simultaneous analysis of DNA content and synthesis, and intracellular and surface epitopes

AU - Jensen, Uffe Birk

AU - Owens, David

AU - Pedersen, Søren

AU - Christensen, Rikke

PY - 2010/8/1

Y1 - 2010/8/1

N2 - 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.

AB - 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.

KW - Animals

KW - Biological Markers

KW - Cell Membrane

KW - Cell Proliferation

KW - DNA

KW - Epitopes

KW - Fixatives

KW - Flow Cytometry

KW - Fluorescence

KW - Intracellular Space

KW - Mice

KW - RNA

KW - Time Factors

KW - Tissue Fixation

KW - Zinc

U2 - 10.1002/cyto.a.20914

DO - 10.1002/cyto.a.20914

M3 - Journal article

C2 - 20653019

SN - 1552-4922

VL - 77

SP - 798

EP - 804

JO - Cytometry. Part A

JF - Cytometry. Part A

IS - 8

ER -