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

Abstract

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 a6 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. ' 2010 International Society for Advancement of Cytometry Key terms stem cell markers; EdU; DNA quantification; long-term storage THE main purpose of chemical fixation is to preserve tissue and subcellular architecture thereby facilitating immunohistochemical detection of epitopes and histochemical detection of chemical substances within cells. Formaldehyde-based fixatives fulfill this requirement to a large extent and have been the preferred compounds for decades in standard pathology procedures. However, a growing demand has emerged for enhanced preservation of proteins and other cellular molecules such as DNA and RNA. In particular, formaldehyde fixation can fragment genomic DNA and hinder quantitative analysis of nucleic acids and generally prohibits PCR amplification of DNA fragments longer than a few hundred base pairs (1). Besides the deleterious effects on DNA, formaldehyde cross-linking alters the tertiary and quaternary structure of proteins and may also mask the secondary structure of globular proteins. Consequently, this can diminish the availability of cellular epitopes to the detriment of immunological-based assays (2). Finally, most formaldehyde fixatives are hazardous and suspected carcinogens, which makes their use dependent on working under a fume hood.As an alternative to formaldehyde-based fixation, zinc salt-based fixation (ZBF), which does not contain any cross-linking agents, is also effective in histopathology procedures (3-6). In fact, studies have shown that RNA, DNA, and proteins are preserved better in ZBF than in neutral buffered formalin (5,6). Furthermore, it was shown that the morphological preservation was comparable to aldehyde-based fixation. Recently, it has been shown that enzymes retain their activity in ZBF-fixed tissue enabling analysis of enzymatic activity in tissue sections (7). However, the use of ZBF has not received a

Cell-Type-Specific Jumonji Histone Demethylase Gene Expression in the Healthy Rat CNS: Detection by a Novel Flow Cytometry Method

“…There is a clear separation in the FSC–SSC plot of mechanically dissociated brain tissue (Figure 1A), representing a visible division between cells and debris. These results are consistent with a report performed on zinc-fixed epithelial cells, showing that ZBF preserves FSC–SSC properties (Jensen et al, 2010). We used DAPI to distinguish between debris and cells with intact nuclei/cells in the cell cycle at the time of fixation (Figure 1B).…”

“…Samples were stored at −20°C overnight or until ready to be used. Consistent with previous reports (Jensen et al, 2010), we have stored samples for several weeks with no detectable loss of cell integrity or immunostaining efficiency (results not shown). To assess the effects of the fixation process on RNA quality, comparisons between fresh and fixed tissues, and fixed/sorted cells were performed.…”

“…Dissociated cells were washed in ice-cold HBSS and pelleted by centrifugation at 350  g for 5 min. Cells were resuspened in a mZBF (0.5% zinc chloride, 0.5% zinc trifluroacetate, 0.05% calcium acetate in 0.1 M Tris–HCL, pH 6.4–6.7) and glycerol (1:1), as previously described (Lykidis et al, 2007; Jensen et al, 2010). Samples were stored at −20°C overnight or until ready to be used.…”

“…However, when endeavoring to isolate nucleic acids from identified and sorted neuron and glial cell populations simultaneously, based on a combination of intracellular and cell surface identification markers, alcohol fixation was ineffective in our studies. We thus turned to a ZBF (zinc-based fixative) which was previously shown to preserve cellular structure, proteins and nucleic acids in histological and cellular studies (Wester et al, 2003; Lykidis et al, 2007; Jensen et al, 2010). Because a mZBF (modified zinc-based fixative) was previously shown to preserve nucleic acids better than the standard zinc fixation methods (Lykidis et al, 2007), we evaluated intracellular, extracellular and nuclear proteins, as well as post-translational modifications to histone tails with mZBF after a mechanical dissociation protocol.…”

Microglial numbers attain adult levels after undergoing a rapid decrease in cell number in the third postnatal week

“…Cells were resuspended in permeabilization buffer (PB, PBS supplemented with 0.1% BSA and 0.2% saponin) for 10 min on ice. After centrifugation, cells were stored at −20°C in a modified zinc-based fixative (0.5% zinc chloride, 0.5% zinc trifluoroacetate, 0.05% calcium acetate in 0.1M Tris-HCL, pH to 6.4–6.7) and glycerol (1:1), as previously described (Jensen et al 2010; Lykidis et al 2007), until further analysis.…”