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

Abstract

Flow cytometry is a useful tool for measuring DNA content and differentiation as expressed by cell surface markers. We have extended this technology to measure simultaneously either surface, cytoplasmic, or nuclear antigens (particularly oncoproteins) with DNA content. Mononuclear blood cells isolated from normal subjects and HL60 leukemic cells were permeabilized and fixed in suspension utilizing 40 pg/ml lysolecithin and 1% paraformaldehyde. A range of lysolecithin concentrations in 1% paraformaldehyde was studied to optimize permeabilization of the antibodies to the cell interior without destroying cell integrity. The optimal concentration (40 pg lysolecithidml) resulted in good cell recovery with a high percentage of cells positive for surface and intracellular antigens. Cells are first stained with fluorescein Flow cytometry is widely used as a method to quantify cell surface antigens and DNA content. It has less application for quantifying intracellular antigens because of the difficulty involved in introducing antibodies into cells without destroying antigenicity and cell integrity. Because of the heterogeneity of cell populations, techniques that enable one to correlate intracellular antigens with cell cycle kinetics could be useful. This is particdarly true for studying oncogene expression correlated with differentiation and cell cycle status.We report a method adapted for flow cytometry that correlates cell cycle kinetics with the amount of intracytoplasmic or intranuclear antigen. The technique utilizes lysolecithin and paraformaldehyde to permeabilize and fix cells simultaneously. With this technique cellular morphology is retained, allowing for the additional correlation of cell surface markers with DNA content. Leukemic cells (HL60 promyelocytic leukemia) were simultaneously stained to correlate DNA isothiocyanate conjugated (FITC) antimyeloperoxidase (an azurophil granule enzyme), or with an anti-c-myc antibody and FITC goat anti-mouse IgG F(ab')2. Cells are then incubated with RNase and stained for DNA content with propidium iodide. Alternatively, cells were stained for the cell surface markers Leu M3, OKM1, or the transferrin receptor and were then fixed and permeabilized and stained with propidium iodide. Using this method, we correlated cytoplasmic, nuclear, or cell surface antigens with cell cycle kinetics. This technique should be useful for studies of cellular differentiation and proliferation.

The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

“…Normal human fibroblast cells were encapsulated in agarose beads and permeabilized with lysolecithin. It was shown previously by Dent et al (1989) that lysolecithin permeabilization permits the entry of high molecular weight proteins into the nucleus. The permeabilized cells were incubated with reaction buffer containing P 32 -labeled dCTP.…”

Purification and characterization of cytolytic and noncytolytic human natural killer cell subsets.

“…Conditions for CMC and preparation of samples for flow cytometric analysis were followed as described (5 Cell Cycle Analysis. Control and sorted cell populations were fixed in 50% (vol/vol) ethanol/PBS, washed twice in PBS, and treated with 0.1% RNase (Sigma) for 30 min at 370C as described (14). Samples were washed twice with PBS and stained with PI (40 pug/ml; Sigma).…”

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

“…Additionally, detergents have been found to alter forward light scatter measurements (4,231. Cell permeabilization with lysolecithin results in extensive cell lysis, leading to low cell preparative yields (6). These effects would be of particular concern in multiparameter flow cytometry studies that include the simultaneous measurement of cell surface markers, intracellular proteins, and/or nuclear DNA.…”

“…Formaldehyde or paraformaldehyde has been used for cell fixation, either with cell membrane permeabilization (4-6,ll-13,17,22), or without cell membrane permeabilization (21). Cell membrane permeabilization agents that have been used with formaldehyde fixation have included methanol (13), acetone (221, Triton X-100 (4,5,11,17,23), or lysolecithin (6). In the few studies that examined paraformaldehyde fixation conditions systematically, different conclusions were reached with regard to optimal fixation concentrations and fixation temperatures for the determination of intracellular antigens.…”