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.     

Quantitative immunofluorescence studies of the serology of Bacillus anthracis spores.

A fluorescein-conjugated antibody against formalin-inactivated spores of Bacillus anthracis Vollum reacted only weakly with a variety of Bacillus species in microfluorometric immunofluorescence assays. A conjugated antibody against spores of B. anthracis Sterne showed little affinity for spores of several B. anthracis isolates including B. anthracis Vollum, indicating that more than one anthrax spore serotype exists.     

Quantitative immunofluorescence, anti-ras p21 antibody specificity, and cellular oncoprotein levels

A general approach to investigating specificity and saturation of antibodies by quantitative immunofluorescence is applied to monoclonal antibodies generated against p21 or ras oligopeptides to quantify ras p21 oncoprotein in cultured cells. Ras 10, a panreactive mouse monoclonal antibody, appears to be a superior probe for detection of p21 in cell extracts or fixed cells because it binds a 21 kD protein on SDS-PAGE/western blots and labels the cytoplasmic membrane in a saturable and competitive manner. RAP-5, a widely used mouse monoclonal antibody generated against an oligopeptide of ras p21, does not recognize p21 in denaturing immunoblots or in immunofluorescence of cultured cells.     

Quantitative immunofluorescence of regulated eps gene expression in single cells of Ralstonia solanacearum.

Ralstonia solanacearum, a phytopathogenic bacterium, uses an environmentally sensitive and complex regulatory network to control expression of multiple virulence genes. Part of this network is an unusual autoregulatory system that produces and senses 3-hydroxypalmitic acid methyl ester. In culture, this autoregulatory system ensures that expression of virulence genes, such as those of the eps operon encoding biosynthesis of the acidic extracellular polysaccharide, occurs only at high cell density (>10(7) cells/ml). To determine if regulation follows a similar pattern within tomato plants, we first developed a quantitative immunofluorescence (QIF) method that measures the relative amount of a target protein within individual bacterial cells. For R. solanacearum, QIF was used to determine the amount of beta-galactosidase protein within wild-type cells containing a stable eps-lacZ reporter allele. When cultured cells were examined to test the method, QIF accurately detected both low and high levels of eps gene expression. QIF analysis of R. solanacearum cells recovered from stems of infected tomato plants showed that expression of eps during pathogenesis was similar to that in culture. These results suggest that there are no special signals or conditions within plants that override or short-circuit the regulatory processes observed in R. solanacearum in culture. Because QIF is a robust, relatively simple procedure that uses generally accessible equipment, it should be useful in many situations where gene expression in single bacterial cells must be determined.     

Quantitative determination of the spatial distribution of pure- and mixed-strain immobilized cells in gel beads by immunofluorescence

A new method was developed to detect and quantify two strains, Lactococcus lactis subsp. lactis biovar. diacetylactis MD and Bifidobacterium longum ATCC 15707, immobilized separately and co-immobilized in gel beads, using specific polyclonal antibodies and confocal laser-scanning microscopy. The establishment of biomass concentration profiles for each strain was measured during colonization of beads using successive pH-controlled batch fermentations. Growth occurred preferentially in 200- and 300-microm peripheral layers of the beads for L. diacetylactis and B. longum, respectively. Repeated-batch cultures with immobilized cells permitted the production of a mixed culture containing a non-competitive strain of bifidobacteria, as a result of immobilized-cell growth and high cell-release activity from the beads. During co-immobilized fermentations, there were no apparent interactions between the strains.     

Quantitative and qualitative immunofluorescence studies of neoplastic cells transfected with a construct encoding p53-EGFP.

The p53 protein is a major regulator of cell cycle progression and apoptosis. We used a p53-enhanced green fluorescent protein (EGFP) construct for transfections into human breast cancer (MCF-7) cells. Cells expressing p53-EGFP showed an increased apoptotic index compared to cells transfected with EGFP alone. Interestingly, apoptotic cells showed localization of p53-EGFP to both nuclei and cytoplasm, whereas non-apoptotic cells usually only showed nuclear localization of p53-EGFP. This result is in agreement with the hypothesis that p53 induces apoptosis by interaction with both nuclear and cytoplasmic targets. Transfected p53-deficient osteosarcoma cells were used for immunofluorescence quantitation. The intensity of immunofluorescence for either p53 or EGFP showed excellent linear correlation to the EGFP autofluorescence, proving that measurements of immunofluorescence intensities can be used for determining endogenous protein levels.     

Intracytoplasmic immunofluorescence in multiple myeloma

A new intracytoplasmic immunofluorescence staining procedure has been investigated to detect and quantify myeloma cells by means of flow cytometry. Freshly harvested bone marrow aspirations from 12 patients with multiple myeloma were treated with collagenase and Triton X-100, and incubated with different specimens of fluoro-isothiocyanate-marked antihuman immunoglobulins. DNA-staining was then done with propidium iodide. Biparametric evaluation in a cytofluorograph 6300A/FC 200 showed a characteristic cluster distribution of normal and pathological immunoglobulin-producing cells. This intracytoplasmic fluorochromic staining procedure may be significant for the specific identification of nonsecretive immunocytomas, which cannot be detected by serodiagnostic methods.     

Detection of Giardia lamblia by immunofluorescence

High-titer immune sera to cysts of Giardia lamblia, produced in guinea pigs, were labeled with fluorescein isothiocyanate. The resulting conjugates were used to detect G. lamblia in stool specimens by fluorescence microscopy. The sera also reacted with cysts of Chilomastix mesnili, but the two organisms could be differentiated by their size.     

Somatic and flagellar immunofluorescence of Salmonella

Caldwell, W. J. (The Child Research Center of Michigan, Detroit, Mich.), C. S. Stulberg, and W. D. Peterson, Jr. Somatic and flagellar immunofluorescence of Salmonella. J. Bacteriol. 92:1177-1187. 1966.-Labeled globulin fractions of flagellar (H) antisera, prepared against 20 frequently occurring Salmonella serotypes belonging to five major somatic (O) groups, were characterized for O and H immunofluorescence and for O and H agglutinin titers against 32 serotypes. The feasibility of immunofluorescent identification of both somatic and flagellar antigens was enhanced by staining formaldehyde-treated organisms in suspension. Relationships between homologous, partial, and unrelated antigen-antibody systems were then analyzed, and a high degree of correlation was shown between the results obtained by the two serological procedures. Flagellar staining was highly specific, and was bright, faint, or inapparent, depending on the relationship between the antigen-antibody systems involved. Somatic staining was also specific, but somewhat more difficult to interpret, because cells in the same preparation might exhibit a mixture of bright, faint, or no fluorescent intensities. Correlation was shown between the percentage of brightly staining cells found in these preparations and the agglutination titers of the comparable antigen-antibody systems. The phenomenon of a "percentage" reaction was unexplained. Absorption studies further confirmed the specificity of reactions. The techniques developed were applied to surveillance of several mouse colonies for the presence of Salmonella. Broth cultures of fecal specimens were treated with formaldehyde and stained in suspension with "polyvalent" labeled antibody reagents. Agreement was found in 97.6% of the instances between results obtained by immunofluorescence and cultural methods. In addition, preliminary evidence indicated the feasibility of presumptive serotyping of Salmonella isolates by immunofluorescence.     

Non-aqueous permanent mounting for immunofluorescence microscopy

It is generally assumed that an aqueous mounting medium is necessary for the preservation of immunofluorescent-labelled microscopical preparations and polyvinyl alcohol-based solutions (e.g. Mowiol) being the most frequently used mounting media; however, both the quality and intensity of the fluorescence signal in most immunolabelled preparations after aqueous mounting slowly diminish with time, and finally, samples become unsuitable for examination. In the present work, we describe a very simple and rapid non-aqueous mounting procedure for cultured cells and tissue sections, which preserves the fluorescent signal in an excellent way after immunodetection or use of other specific labelling methods. It is based on the current histological protocol in which, after fluorescence labelling, preparations are dehydrated in ethanol, cleared in xylene and mounted in DePeX. Using this non-aqueous mounting medium, the fluorescent signal remains high and stable, allowing a suitable and permanent preservation of labelled and counterstained microscopical preparations.     

Retardation of immunofluorescence fading during microscopy

Polyvinyl alcohol (PVA) mounting medium containing paraphenylenediamine (PPD), n-propyl gallate (NPG), or 1,4-diazobicyclo(2,2,2)-octane (DABCO) was compared with PVA alone or buffered glycerol with regard to capacity for preservation of immunofluorescence preparations. The results were based on staining of an artificial substrate with homogeneous antigen distribution followed by microphotometric determination of the initial light emission from bound fluorescein isothiocyanate (FITC)-labeled antibody and the subsequent fluorescence fading during 3-min exposure to blue excitation light. At a concentration of 0.2-2.0 g/liter and 6 g/liter, respectively, PPD and NPG were shown to effectively retard fluorescence fading without notably decreasing the initial emission intensity; two requisites were that the modified PVA used must be rather fresh and that the mounted preparations be examined within a few days. Although addition of DABCO (6 g/liter) afforded a mounting medium that tolerated storage before use better, but both PPD and NPG were more advantageous in practice. The retarding effect of PPD on fading of FITC emission was confirmed by performance testing on human tissue sections. Remounting in PVA alone is recommended for prolonged storage of sections that have been mounted in PVA modified with one of the above-mentioned compounds.