Reactions of seven basic fluorochromes with unfixed cells obtained from the salivary glands of the dipteran fly Megaselia scalaris Loew (Phoridae)

Summary Seven basic fluorochromes with varying specificities were used to stain the large squamous epithelial cells isolated from the larval salivary glands of Megaselia scalaris (Phoridae). Although the EDTA-based method selected for isolating the cells produced permeabilization and a loss of viability of the cells, consistent results were obtained with the various fluorochromes. The classical pattern of green nuclear and red cytoplasmic fluorescence observed in cells stained with acridine orange could be changed to green cytoplasmic and red nuclear fluorescence by pretreatment with RNase. The predominantly cytoplasmic and nucleolar fluorescence obtained with pyronine Y could be changed to mainly nuclear fluorescence by RNase pretreatment. The other five fluorochromes tested were not affected appreciably by extraction with RNase. Quinacrine mustard, dicarbocyanine (DiOC₆(3)), and rhodamine 123 produced primarily cytoplasmic and nucleolar fluorescence, while nile red revealed mainly cytoplasmic lipid droplets. Phosphine 3R initially stained lipid droplets but very rapidly redistributed throughout the cytoplasm and nucleus. Because of their large size, flatness, and content of histochemically demonstrable components, the cells of Megaselia are especially appropriate for use as optical objects or controls in various studies. New methods of isolating the cells, however, will be needed to prevent permeabilization and loss of viability of the cells.     

Romanowsky-type staining: Enzymatic analysis of nuclear metachromasia in formalin-fixed paraffin sections

The red color of nuclei produced in formol-fixed paraffin sections stained with toluidine blue has been investigated by using deoxyribonuclease (DNase), ribonuclease (RNase) and 0.1 M Tris buffer. The action of DNase on formol-fixed material is not fully reliable, but clear-cut when positive. Nuclear basophilia and metachromasia is removed, nucleolar and cytoplasmic RNA is preserved. The picture produced by RNase depends to some extent on the concentration and acidity of the toluidine blue used for subsequent staining. Cytoplasmic RNA is always removed, while the red stain in nuclei usually remains intact. With 0.1% toluidine blue in 1% acetic acid, a nuclear color change from red to pale green is observed. Using this same staining solution, it can be shown that 0.1 M Tris buffer (overnight extraction at 37° C) will remove cytoplasmic RNA but leave intact the nuclear material that stains red. A red to green shift can subsequently be produced by RNase. From this it is deduced that there is a chromatin-associated nuclear RNA fraction which can be removed by the enzyme, but is stable to the buffer solution.     

Automatic cell identification and enrichment in lung cancer. III. Light scatter and two fluorescence parameters.

Two fluorescence parameters and size are used in a flow through system to enrich sputum specimens for cancer cells. Human cells in sputum which are stained with acridine orange show a characteristic distribution of red and green fluorescence from which cancer cells can be localized. The peak enrichment is obtained by selectively sorting cells with the largest values of red and green fluorescence. Cancer cells located in other distribution regions having smaller fluorescence intensities show progressively diminished nuclear and cytoplasmic tinctorial features by Papanicolaou stain, consistent with the decreased intensity of red and green fluorescence.     

Determination of Cryptosporidium parvum oocyst viability by fluorescence in situ hybridization using a ribosomal RNA-directed probe

AIMS: Fluorescence in situ hybridization (FISH) has been proposed for species-specific detection, and viability determination of Cryptosporidium parvum oocysts. FISH-based viability determination depends on rRNA decay after loss of viability. We examined the effects of RNase(s) and RNase inhibitors on FISH of C. parvum. METHODS AND RESULTS: FISH was performed using a 5'-Texas red-labelled DNA oligonucleotide probe at 1 pM microl(-1). Intact and heat-permeabilized oocysts were treated with 1-100 microg ml(-1) RNase. FISH of intact oocysts appeared unaffected by exogenous RNase if this was neutralized before permeabilization. FISH fluorescence of heat-killed oocysts stored in phosphate-buffered saline at room temperature decayed by 1/2 after 55 h, but remained detectable after 6 days. Addition of vanadyl ribonucleoside complex (VRC) extended rRNA half-life of heat-permeabilized oocysts to 155 h. CONCLUSIONS: Extended rRNA half-life may result in viability overestimation using FISH. RNase pretreatment before FISH is recommended to destroy residual rRNA in recently killed oocysts. Incorporation of 1-10 mM l(-1) VRC before FISH permeabilization steps should neutralize RNase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Elimination of FISH fluorescence of nonviable C. parvum is desirable. Use of RNase and VRC is suggested to reduce numbers of false-positive 'viable' oocysts.     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange.

Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and muscopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracelluar dye contents as low as 5 X 10(-16) mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.     

Cytochemical significance of acridine orange staining of human plasmodia with some comments on double-stranded DNA

Summary Air-dried blood smears and erythrocyte suspension from patients infected with Plasmodium falciparum, stained under optimal conditions with acridine orange, permit easy detection of plasmodia with fluorescence microscopy together with a clear cytochemical colour differentiation of nuclear DNA (green or green-yellow) and cytoplasmic RNA (orange-red fluorescence). Judging from fluorescence characteristics of nuclei (DNase sensitive and RNase resistant green or green-yellow), the plasmodial DNA appears to be double-stranded.     

Fluorescence fading and stabilization in cytofluorometry

The factors affecting fluorescence fading in cytofluorometry were investigated using different kinds of nuclear staining, mounting media, and procedure of specimen preparation. Acceleration of fluorescence fading was observed in smear specimens treated with RNase, trypsin, or hypotonic solution before pararosaniline Feulgen nuclear staining. Similar effect was found for other DNA-stainings such as "33258 Hoechst" and Feulgen reactions with different Schiff-type dyes, such as acriflavine-SO2 and cresyl-violet-SO2, when chemically pure DNA was used. Fluorescence decay was rapid for all fluorochromes examined, when glycerin or buffer solution was used as mounting medium. Marked stabilization of fluorescence emission was induced in specimen mounted in non-fluorescent resin, Entellan (Merck), after post-staining fixation with absolute methanol for all tested fluorochromes. The same treatment induced almost complete fluorescence stabilization of fluorescein isothiocyanate (FITC); no detectable fluorescence fading was observed in a specimen stained with indirect immunofluorescence reaction using anti-UV-DNA antibody, during storage for 2 years at room temperature without special protection against light. These observations suggest that factors which bring about conformational stability of macromolecule-dye complexes generally induce fluorescence stabilization.     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange

Summary Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and mucopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracellular dye contents as low as 5×10^–16 mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.     

Nile red: a selective fluorescent stain for intracellular lipid droplets

We report that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry. The specificity of the dye for lipid droplets was assessed on cultured aortic smooth muscle cells and on cultured peritoneal macrophages that were incubated with acetylated low density lipoprotein to induce cytoplasmic lipid overloading. Better selectivity for cytoplasmic lipid droplets was obtained when the cells were viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) rather than red fluorescence (excitation, 515-560 nm; emission, greater than 590 nm). Nile red-stained, lipid droplet-filled macrophages exhibited greater fluorescence intensity than did nile red-stained control macrophages, and the two cell populations could be differentiated and analyzed by flow cytofluorometry. Such analyses could be performed with either yellow-gold or red fluorescence, but when few lipid droplets per cell were present, the yellow-gold fluorescence was more discriminating. Nile red exhibits properties of a near-ideal lysochrome. It is strongly fluorescent, but only in the presence of a hydrophobic environment. The dye is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution. Nile red can be applied to cells in an aqueous medium, and it does not dissolve the lipids it is supposed to reveal.     

The staining behavior of oocyte cytoplasm with the alcoholic Feulgen variant and with methyl green

Summary A variant of the Feulgen reaction which has been proposed as a method for demonstrating cytoplasmic DNA in oocytes has been tested on ovarian material from a variety of species. While Schiff positive staining was developed, this was not removable by pretreatment with DNase and could be reproduced by using oxidants used in the pseudoplasmal reaction. This method was not considered useful for demonstrating cytoplasmic DNA.When chloroform extracted solutions of methyl green were used to stain ovaries, cytoplasmic staining identical in pattern to that obtained with other basic dyes was observed. The cytoplasmic staining was prevented by pretreatment of sections with RNase, but was not affected by DNase pretreatment. In somatic cells with high concentrations of cytoplasmic RNA, only nuclear staining was observed. This nuclear staining was labile to DNase but not to RNase.This work was supported by U.S. Public Health Service grants GM-10003-03 and K-3-6176-03.Contribution number 376 of the Bermuda Biological Station.     

Flow cytometric prescreening of cervical smears.

One-parameter (nuclear DNA) and two-parameter (nuclear DNA and protein or cellular light scatter) measurements of cervical smears were performed using an ICP 11 and a cytofluorograf 4800 respectively. A total of about 1000 cases was analyzed. For the estimation of nuclear DNA alone two fluorochromes were tested (ethidium bromide (EB) and mithramycin (MMC)) combined with three different methods of cell preparation. For the two-parameter measurements cells were double stained with EB and fluorescein isothiocyanate (FITC). Red fluorescence (EB) versus green fluorescence (FITC) or red fluorescence versus scatter were recorded. A computer analysis of the one-parameter histograms was performed using discriminant analysis and the results were compared with the cytodiagnosis of microscopic specimens stained with the Papanicolaou technique. The error rates of the flow cytometric (FCM) data were as follows: (a) standard EB staining, 11% false negative, 26% false positive, 6% unsatisfactory results; (b) pepsination of vital cells and EB staining, 12% false negative, 14% false positive and 4% unsatisfactory results; (c) MMC staining, 10% false negative, 65% false positive and 5% unsatisfactory results. Our two-parameter measurements prove that, as confirmed by cell sorting, red fluorescence versus scatter allows separation of at least three subpopulations in most analyzed samples: (a) anucleated cells; (b) leukocytes; and (c) intermediate and superficial cells.     

Fluorescence fading and stabilization in cytofluorometry

Summary The factors affecting fluorescence fading in cytofluorometry were investigated using different kinds of nuclear staining, mounting media, and procedure of specimen preparation. Acceleration of fluorescence fading was observed in smear specimens treated with RNase, trypsin, or hypotonic solution before pararosaniline Feulgen nuclear staining. Similar effect was found for other DNA-stainings such as 33258 Hoechst and Feulgen reactions with different Schiff-type dyes, such as acriflavine-SO₂ and cresylviolet-SO₂, when chemically pure DNA was used. Fluorescence decay was rapid for all fluorochromes examined, when glycerin or buffer solution was used as mounting medium. Marked stabilization of fluorescence emission was induced in specimen mounted in non-fluorescent resin, Entellan (Merck), after post-staining fixation with absolute methanol for all tested fluorochromes. The same treatment induced almost complete fluorescence stabilization of fluorescein isothiocyanate (FITC); no detectable fluorescence fading was observed in a specimen stained with indirect immunofluorescence reaction using anti-UV-DNA antibody, during storage for 2 years at room temperature without special protection against light. These observations suggest that factors which bring about conformational stability of macromoleculedye complexes generally induce fluorescence stabilization.     

Pyronin Y as a fluorescent stain for paraffin sections

Pyronin Y has long been used, in combination with other dyes such as Methyl Green, as a differential stain for nucleic acids in paraffin tissue sections. It also forms fluorescent complexes with double-stranded nucleic acids, especially RNA, enabling semi-quantitative analysis of cellular RNA in flow cytometry. However, the possibility of using pyronin Y as a fluorescent stain for paraffin tissue sections has rarely been investigated. We herein report that in sections stained with Methyl Green–pyronin Y, red blood cells, elastic fibre of blood vessels, zymogen granules of pancreatic acinar cells, surface membrane of heptocytes and kidney tubular cells showed strikingly strong green and/or red fluorescence, while the nuclei of cells appeared non-fluorescent. The use of confocal laser-scanning microscope greatly improved the resolution and selectivity of the fluorescent images. Staining with pyronin Y alone gave similar results in terms of fluorescence properties of the specimens. Pretreatment of paraffin sections with RNase significantly reduced cytoplasmic pyronin Y staining as judged by transmission light microscopy, but it had little effect on the fluorescence intensity of red blood cells, elastic fibres and zymogenbreak granules.     

A fluorescence‐activated cell sorting‐based strategy for rapid isolation of high‐lipid Chlamydomonas mutants

There is significant interest in farming algae for the direct production of biofuels and valuable lipids. Chlamydomonas reinhardtii is the leading model system for studying lipid metabolism in green algae, but current methods for isolating mutants of this organism with a perturbed lipid content are slow and tedious. Here, we present the Chlamydomonas high‐lipid sorting (CHiLiS) strategy, which enables enrichment of high‐lipid mutants by fluorescence‐activated cell sorting (FACS) of pooled mutants stained with the lipid‐sensitive dye Nile Red. This method only takes 5 weeks from mutagenesis to mutant isolation. We developed a staining protocol that allows quantification of lipid content while preserving cell viability. We improved separation of high‐lipid mutants from the wild type by using each cell's chlorophyll fluorescence as an internal control. We initially demonstrated 20‐fold enrichment of the known high‐lipid mutant sta1 from a mixture of sta1 and wild‐type cells. We then applied CHiLiS to sort thousands of high‐lipid cells from a pool of about 60 000 mutants. Flow cytometry analysis of 24 individual mutants isolated by this approach revealed that about 50% showed a reproducible high‐lipid phenotype. We further characterized nine of the mutants with the highest lipid content by flame ionization detection and mass spectrometry lipidomics. All mutants analyzed had a higher triacylglycerol content and perturbed whole‐cell fatty acid composition. One arbitrarily chosen mutant was evaluated by microscopy, revealing larger lipid droplets than the wild type. The unprecedented throughput of CHiLiS opens the door to a systems‐level understanding of green algal lipid biology by enabling genome‐saturating isolation of mutants in key genes.     

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

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 micrograms/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 micrograms lysolecithin/ml) resulted in good cell recovery with a high percentage of cells positive for surface and intracellular antigens. Cells are first stained with fluorescein 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.     

Automatic cell identification and enrichment in lung cancer. II. Acridine orange for cell sorting of sputum.

Fluorescence spectra were obtained from cells from sputum and pleural effusions stained with different fluorescent dyes and fixed by alternate methods. The spectra were referenced to a standard allowing for fluorescence comparisons of unstained and stained cells under various conditions. The metachromasia of acridine orange-stained cells offers nuclear/cytoplasmic differentiation in a single stain; mithramycin and propidium iodide do not. Unstained cells have an appreciable amount of green (546 nm) fluorescence, as does Carbowax in Saccomanno's preservative. Cytoplasm stained with acidine orange also has appreciable green fluorescence. Consequently, cells with much cytoplasm have high total fluorescence. Cytoplasmic fluorescence is negligible with mithramycin or propidium iodide. The metachromasia of acridine orange-stained cells is altered by alcohol and Carbowax levels in fixatives, keeping other factors constant.     

Autologous lymphocyte-monocyte co-culture increases NMR-visible and cytoplasmic lipids in the absence of increased markers of lymphocyte activation.

Alterations in nuclear magnetic resonance (NMR)-visible lipid, morphometric lipid volume fraction, distribution of subcellular lipid droplets and activation antigen expression were examined in human peripheral blood lymphocytes, activated using phorbol myristate acetate (PMA) and ionomycin or by co-culture with autologous monocytes. PMA/Ionomycin treatment caused significant time-dependent increases in mobile lipid and in oil red O-positive lipid droplets that were accompanied by lymphocyte proliferation and increases in activation antigens, CD25, CD69 and CD71. Co-culture of lymphocytes and monocytes also induced significant increases in NMR-visible lipid signals and cytoplasmic lipid droplets, but in contrast, no correspondent increases in activation antigens were observed. Strong correlations were observed between the intensity of the NMR signal and the percentage of total cells containing lipid droplets (r=0.95) and the morphometric lymphocyte lipid volume fraction (r=0.80), indicating that the droplets were the source of the mobile lipid signal. Lipid droplets in PMA/Ionomycin-treated cells were evenly distributed throughout the population, but in co-cultures, only lymphocytes in close proximity to monocytes with lipid droplets contained oil red O-positive lipid. This data shows that the NMR-visible mobile lipid signal observed in lymphocytes co-cultured with monocytes is not directly dependent on either proliferation or the upregulation of activation antigens, similar to the previously observed response of T cells exposed to antibodies to the T cell receptor.     

Microfluorometric investigations of chromatin structure. I. Evaluation of nine DNA-specific fluorochromes as probes of chromatin organization

The ability of the highly condensed chromatin of small thymocyte nuclei and the more loosely organized chromatin of hepatocyte nuclei to interact with nine DNA-specific fluorochromes was assessed by microfluorometry. Although the results obtained with five of the fluorochromes - mithramycin, 7-aminoactinomycin D, Hoechst 33258, DAPI, and propidium iodide - were found to be virtually unaffected by differences in the degree of condensation of the chromatin, the values obtained with the remaining fluorochromes - proflavine, quinacrine mustard, berberine sulfate, and pyronin Y - appeared to be affected significantly by organizational differences of the chromatin. All of the latter "structural probes," except quinacrine mustard, produced fluorescence values which were higher in the 2c nuclei of hepatocytes than in the nuclei of small thymocytes. Quinacrine mustard yielded higher values in thymocyte nuclei; and in the hepatocyte polyploid series (2, 4, and 8c), it did not produce the expected multiples of the 2c value. Pretreatment of the two types of nuclei with RNase affected their total fluorescence in unpredictable ways. While RNase extraction lessened the differences between thymocyte and 2c hepatocyte nuclei stained with propidium iodide, Hoechst 33258, proflavine, and berberine sulfate, it increased the differences between nuclei stained with mithramycin, quinacrine mustard, pyronin Y, and 7-aminoactinomycin D. The ability of RNA-depleted chromatin to interact with various types of fluorochromes might be a useful parameter in subsequent studies of chromatin organization.     

Microspectrofluorometry of ultraviolet-inducible fluorescence in supravitally-stained ascites tumour cells

Synopsis Ultraviolet irradiation of tumour cells (Ehrlich tetraploid ascites tumour of mice, TO strain), supravitally stained with thiazine dyes (Azure II, Azure A, Methylene Blue, Toluidine Blue) or an oxazine dye (Brilliant Cresyl Blue), induces blue fluorescence in cytoplasmic bodies believed to be lipid droplets or lysosome-like bodies. Microspectrofluorometry of the inducible fluorescence in Ehrlich tumour cells gives bimodal excitation (340/394 nm) and emission (443/700 nm) curves.     

Effects of alpha-amanitin on the fine structure of adrenal fasciculata cells in the young rat

The administration of 0.2 micrograms/g/bw of alpha-amanitin to approximately 20 g rats provoked the following nuclear modifications in rat adrenal fasciculata cells at 60 min: chromatin condensation, nucleolar fragmentation, increase in the number of PCG and clumping of ICG in the center of the nucleus. At longer time intervals (2.5 and 4.5 hr) these alterations were more evident, but at 24 hr the nuclear structure was back to normal with the exception of a persistent increased number of PCG. After injection of 0.75 micrograms/g/bw and at 2.5 hr, there was pulverization of condensed chromatin, fragmentation and partial segregation of the nucleolus with increased density of the fibrillar component. Cytoplasmic alterations were severe including cap-shaped mitochondria with electron-dense matrix surrounding lipid droplets, reduced endoplasmic reticulum of vesicular type and clusters of microvesicles with dense content in the Golgi trans-area. At 24 hr, the nuclear and cytoplasmic morphology returned to normal. These findings are interpreted as the morphological counterpart of a disturbance of extranucleolar and nucleolar RNA synthesis, as well as of lipid and lipoprotein metabolism, brought about by the drug.