Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

doi:10.1002/CYTO.990130414

Paper

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

Published 1992 · A. Pollice, J. McCoy, Stanley E. Shackney

Cytometry

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117

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3

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Abstract

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p less than 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p less than 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37 degrees C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors.

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Sequential paraformaldehyde and methanol fixation is suitable for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins, with minimal effects on cell morphology and light scatter properties.

PopulationOlder adults (50-71 years)

Sample size24

MethodsObservational

OutcomesBody Mass Index projections

ResultsSocial networks mitigate obesity in older groups.

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Paper

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

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References

P-glycoprotein expression in plasma-cell myeloma is associated with resistance to VAD.

P-glycoprotein expression in plasma-cell myeloma is associated with resistance to VAD therapy, indicating diploid cells may play a role in the disease process.

Simultaneous measurement of progesterone receptors and DNA indices by flow cytometry: characterization of an assay in breast cancer cell lines.

This study developed a flow cytometry method to simultaneously measure progesterone receptors and DNA indices in breast cancer cell lines, revealing increased PR levels in T47D cells and no specific binding in MDA-231 cells.

Simultaneous paired analysis by flow cytometry of surface markers, cytoplasmic antigens, or oncogene expression with DNA content.

This study developed a flow cytometry technique that allows simultaneous measurement of surface, cytoplasmic, or nuclear antigens with DNA content, potentially aiding in studies of cellular differentiation and proliferation.

Flow-cytometric detection of terminal deoxynucleotidyl transferase and other intracellular antigens in combination with membrane antigens in acute lymphatic leukemias

A new fixation procedure enables rapid and quantitative flow cytometric analysis of intracellular antigens in acute lymphatic leukemia, enabling rapid and quantitative immunodiagnosis.

Correlated flow cytometric analysis of H-ras p21 and nuclear DNA in multiple myeloma.

High H-ras p21 levels in aneuploid multiple myeloma cells suggest a role for the H-ras oncogene in the disease, with shorter survival in patients with high p21 levels.

Citations

Approaches for the Inactivation of Yersinia pestis.

This study provides examples of dose-dependent kill curves for commonly used inactivation methods against Yersinia pestis, enabling researchers to develop their own validated inactivation protocols.

Immunocytochemical Reactivity Comparison between Formalin-Fixed and Liquid-Based Cytology-Fixed Specimens

ICC using liquid-based cytology-fixed samples shows the same immunoreactivity as formalin-fixed samples, with the percentage of positive cells increasing or decreasing based on the type of fixing solution and antigen concentration.

High-throughput RNA sequencing of paraformaldehyde-fixed single cells

FD-seq is a high-throughput method for RNA sequencing of paraformaldehyde-fixed single cells, enabling phenotypic and transcriptomic integration in rare cell subpopulations and pathogenic samples.

Efficacy of the Antigenicity-Retaining Ability of Fixative Solutions for Liquid-Based Cytology: Immunocytochemistry of Long-Term Storage

Long-term storage of cell membrane antigens at room temperature is suitable, but unsuitable for intranuclear antigens.

Unfavorable Effects of Fixatives on the Fluorescence Intensity and Biological Functions of Fluorescent Proteins in HEK293T Cells and Transgenic Mice

Fixatives can weaken or eliminate fluorescent protein signals, affecting their biological functions and requiring careful application in experiments.