Quantitative immunofluorescence in single Saccharomyces cerevisiae cells.
We have developed a staining procedure that allows the simultaneous determination of intracellular amounts of DNA and an antigen in Saccharomyces cerevisiae with a single laser flow cytometer. The antigen, beta-galactosidase from a cloned lacZ gene, is inducible and is detected with an indirect immunofluorescent stain. Cell preparation procedures, specifically cell fixation and cell wall removal, have significant effects on measured levels of immunofluorescence and have been optimized to prevent cell loss and maximize immunofluorescence. Average immunofluorescent levels of cell populations expressing different levels of beta-galactosidase show excellent correlation with measurements of average beta-galactosidase activity per cell based on cleavage of o-nitrophenyl-beta-D-galactopyranoside. Experiments with yeast populations containing various numbers of copies of the cloned gene indicate that the relationship between immunofluorescence and antigen content also holds at the single-cell level. Correlated measurements of DNA and beta-galactosidase content on a single-cell level permit the investigation of cellular enzyme content as a function of cell cycle position under various conditions. The procedure can be easily modified to detect other antigens by changing the primary antibody used.
Eitzman
P D
PD
Institute for Advanced Studies in Biological Process Technology, University of Minnesota, St. Paul 55108.
Hendrick
J L
JL
Srienc
F
F
D004269:DNA, Bacterial / Q000032:analysis*; D005404:Fixatives; D005434:Flow Cytometry / Q000379:methods*; D005696:Galactosidases / Q000032:analysis*; D007150:Immunohistochemistry / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000032:analysis* / Q000378:metabolism; D001616:beta-Galactosidase / Q000032:analysis*
D004269:DNA, Bacterial; D005404:Fixatives; D005696:Galactosidases; D001616:beta-Galactosidase