Methodologies for the preservation of proliferation associated antigens PCNA, p120, and p105 in tumor cell lines for use in flow cytometry.
The retention of antigen expression of PCNA, p120, and p105 in two tumor cell lines (MOLT-4 and MDA-MD-175-VII) under various conditions of fixation was investigated using flow cytometric analysis. Four currently utilized procedures for fixation/permeabilization of intracellular antigens were compared for their ability to stain the nuclear antigens. A procedure using a brief incubation in a solution of lysolecithin in paraformaldehyde followed by fixation in ice-cold methanol prior to antibody staining was selected to evaluate reagent protocols aimed at preserving antigen expression. Holding samples overnight at 4 degrees C in 2.5% fetal bovine serum after the lysolecithin/paraformaldehyde and methanol fixation steps prior to staining with monoclonal antibodies resulted in no decline in the percentage of cells positively stained for all three markers with little decrease in intensity of fluorescence and no increase in DNA coefficient of variation (c.v.). Fixed/permeabilized MOLT-4 cells held longer than 24 h before staining were lower in PCNA fluorescence than freshly stained cells; holding samples of either cell line longer than 48 h resulted in decreased PCNA and p120 staining. Prefixing and holding cells in 50% methanol or 50% ethanol overnight before processing and staining severely depressed PCNA and p120 fluorescence. Prefixing either cell line in a range of concentrations (0.25-1.0%) of paraformaldehyde also resulted in reduced intensity of PCNA and p120 fluorescence along with increased DNA c.v. P105 staining appeared to be relatively unaffected by all prefixation/storage conditions tested, except for a decline of fluorescence when MDA cells were prefixed in 50% ethanol. Cells cryopreserved in liquid nitrogen for 1 week before processing showed < 5% loss of PCNA and p120 fluorescence compared to freshly processed cells, but p105 fluorescence dropped 29% in cryopreserved MDA cells. These results underscore the fact that specific protocols for the fixation and storage of biological samples prior to staining and analysis must be determined for the specific nuclear antigen marker under investigation.
Mikulka
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Coulter Technology Center, Miami, Florida 33116-9015.
Bolton
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D002453:Cell Cycle; D002463:Cell Membrane Permeability; D015925:Cryopreservation; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D009687:Nuclear Proteins / Q000032:analysis*; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D016707:Tissue Fixation / Q000379:methods*; D014407:Tumor Cells, Cultured / Q000737:chemistry*
D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen