Immunofluorescent flow cytometry inN dimensions

The problem of the simultaneous use in flow cytometry ofN>2 antibodies in conjunction with two fluorochromes was investigated. Theoretical analysis led to a labeling procedure and reconstruction formula that allowN-dimensional labeling distributions to be obtained from two-dimensional fluorescence distributions. The general problem ofM≥2 fluorochromes andN>M antibodies was shown to be reducible to the case of two fluorochromes. The method was tested by a triple labeling analysis of murine thymocytes.     

Monofunctional near-infrared fluorochromes for imaging applications

In this report, the development of a new class of monocarboxylate functionalized cyanine derivatives using improved synthetic procedures is detailed. The employed synthetic strategy relies on efficient nucleophilic attack of alkyl-thiols on cyanine dyes bearing chloro-substituted polymethinic linkers. Monocarboxylate derivatized fluorochromes (CyTE dyes) can be prepared in one step in greater than 90% yield without the need for additional purification. Several of the fluorochromes synthesized by this route show no tendency to aggregate in aqueous solution and have excitation and emission maxima greater than 800 nm. The potential utility of the CyTE fluorochromes was demonstrated through direct labeling of phage displaying a vascular cellular adhesion molecule-1 (VCAM-1) targeting peptide. Endothelial cell internalization of the VCAM-1 targeted phage was monitored via near-infrared fluorescence microscopy.     

A comparison of the highly selective fluorescence staining of fungi in tissue sections with Uvitex 2B and Calcofluor White M2R

Summary The selective fluorescence staining of two fungi,Candida albicans andBlastomyces dermatitides, with Uvitex 2B and Calcofluor White M2R was studied in deparaffinized and frozen sections of mouse kidney and lung. Both fluorochromes emitted maximally at about 430nm, independent of the mounting media (Kaiser's gelatin or Entellan). In addition to fungi, both fluorochromes also stained elastic fibres. The fluorescence intensity remained unchanged after storage of sections for more than 6 months in conventional slide boxes. the two fluorochromes showed the following differences: Calcofluor faded 1.25 times faster than Uvitex when illuminated with ultraviolet light. Calcofluor showed a greater affinity for tissues in general, and red cells and renal tubular casts in particular. Counterstaining of deparaffinized sections with Hemalum and Eosin reduced the fungi fluorescence and suppressed the general background fluorescence. However, it led to an intensification of Eosin staining and the fluorescence of red cells in Calcofluorstained sections but not in Uvitex-stained ones. Similarly, the background fluorescence in frozen sections was reduced by Evans Blue, although elastic fibres still fluoresced after staining with Calcofluor. The degree of staining selectivity, and thus the contrast produced within a histological specimen, was greater with Uvitex 2B than with Calcofluor White M2R.     

A new method for rapid and sensitive detection of bromodeoxyuridine in DNA-replicating cells

A new flow cytometric technique, involving differential fluorescence analysis of two DNA-binding fluorochromes, was used to quantify cellular incorporation of the base analog, bromodeoxyuridine (BrdU), into DNA over short time periods. During analysis of stained cells, the blue fluorescence signal of Hoechst 33342, which is quenched by BrdU-substituted DNA, was subtracted, on a cell by cell basis, from the green-yellow fluorescence signal of mithramycin, which remained stoichiometric to cellular DNA content. Bivariate contour profiles obtained for CHO cells pulse-labeled for 30 min showed that fluorescence quenching of Hoechst 33342 in BrdU-labeled, S phase cells produced fluorescence difference signals that were significantly greater than the difference signals from G1 and G2 + M phase cells. Analysis of L1210 cells demonstrated that the amount of BrdU detected was proportional to the length of the labeling period. The novel technique is simple, rapid, and mild; it produces minimal cell loss and does not significantly affect cellular moieties such as DNA, chromatin, or RNA.     

Flow cytometric characterization of normal and variant cells with monoclonal antibodies specific for glycophorin A

Quantitative immunofluorescence measurements were performed on erythrocytes labeled with monoclonal antibodies to glycophorin A (GPA) to assess the level of binding of these antibodies to normal and variant cell types. The seven antibodies used in this study include two that bind preferentially to the M form of GPA, three that bind preferentially to the N form, and two that bind equally well to both. Flow cytometric analysis of mixtures of cells differing in M,N type showed binding specificities of greater than 100-fold for most of the antibodies, and showed that three antibodies bind cell-bound GPA with an affinity of approximately 10(9) M(-1). These data also showed that the level of expression of GPA varies by less than 10% from cell to cell and from individual to individual. Flow measurements were also done on human erythrocytes with the following variant forms of glycophorin: Mc, Mg, Mk, En(F), En(UK), Mi-I, Mi-II, Mi-III, S-s-U-, Tn+, and St(a+). Other cell types analyzed included erythrocytes from chimpanzee, rhesus, African green, and capuchin monkeys, and cells from the human erythroleukemia cell line, K562. Flow analysis with our seven antibodies showed these cell types have distinctive labeling patterns consistent with the known or inferred altered glycophorins presented on these cells. In most cases, variant alleles were expressed at normal levels. Our results support other observations that the variants En(UK) and St(a+) contain hybrid GPA-GPB proteins, and suggest that their level of expression is largely determined by the 3' end of the hybrid genes.     

Use of tetrameric antibody complexes to stain cells for flow cytometry

Bifunctional tetrameric complexes of monoclonal antibodies were used to stain cells for flow cytometry. These complexes consist of two different mouse monoclonal IgG1 antibodies (one with specificity for a cell surface antigen, the other with specificity for a fluorochrome) cross-linked by two molecules of a monoclonal rat anti-mouse IgG1. The use of this immunological approach to cross-link fluorochromes to cell surface antigens was studied with tetrameric complexes containing Leu-3a or Leu-2a antibodies and monoclonal antibodies specific for the fluorochromes B- and R-phycoerythrin. The ability of such cyclic immune complexes to stain T-cell subset antigens on human peripheral blood lymphocytes was demonstrated in single and double-staining experiments. The results demonstrate that tetrameric antibody complexes provide a simple and efficient alternative to covalently labeled antibodies for the flow cytofluorimetric analysis of cell-surface antigens.     

Detection of endogenous and antibody-conjugated alkaline phosphatase with ELF-97 phosphate in multicolor flow cytometry applications

BACKGROUND: The fluorogenic alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF(R)-97 phosphate, for Enzyme-Labeled Fluorescence) has been used primarily in microscope-based imaging applications to detect endogenous AP activity, antigens and various ligands in cells and tissues, and nucleic acid hybridization. In a previous study, we demonstrated the applicability of ELF-97 phosphate for detecting endogenous AP activity by flow cytometry. In this study, we show that the spectral characteristics and high signal-to-noise ratio provided by the ELF-97 phosphate make it a useful label for immunodetection via flow cytometry. It can be combined with a variety of other fluorochromes for multiparametric flow cytometry analysis of both endogenous AP activity and intracellular and extracellular immunolabeling with AP-conjugated antibodies. METHODS: ELF-97 phosphate detection of endogenous AP activity in UMR-106 rat osteosarcoma cells was combined with intracellular antigen detection using Oregon Green 488 dye-conjugated secondary antibodies and DNA content analysis using propidium iodide (PI) or 7-aminoactinomycin D (7-AAD). ELF-97 phosphate detection of endogenous AP was also tested for spectral compatibility with a variety of other commonly used fluorochromes. ELF-97 phosphate was then used to directly label intracellular antigens via AP-conjugated antibodies, again combined with the analysis of DNA content using PI and 7-AAD. ELF-97 phosphate was also used to directly detect extracellular antigens. It was combined with Oregon Green 488 dye, phycoerythrin (PE), and PE-Cy5 dye-labeled antibodies for simultaneous four-color analysis. All samples were analyzed on a dual-beam flow cytometer, with UV excitation of the ELF-97 alcohol reaction product. RESULTS: Application of the ELF-97 phosphate to detect AP was found to be compatible with immunodetection and DNA staining techniques. It was also spectrally compatible with a variety of other fluorochromes. Endogenous AP activity could be detected simultaneously with both intracellular antigens labeled using Oregon Green 488 dye, PE, Cy5 dye and Alexa Fluor 568 dye-conjugated antibodies, and DNA content analysis with PI or 7-AAD. This multiparametric assay accurately delineated the distribution of AP in cycling cells and was able to identify cell subsets with varying endogenous AP levels. The ELF-97 alcohol reaction product was found to be an effective label for intracellular antigen immunolabeling with AP-conjugated reagents, and could also be combined with PI and 7-AAD. ELF-97 phosphate was also found to be a useful label for extracellular antigen immunolabeling with AP conjugates, and was compatible with Oregon Green 488 dye, PE, and PE-Cy5 dye-labeled antibodies for four-color surface labeling with minimal spectral overlap and color compensation. CONCLUSIONS: ELF-97 phosphate was shown to be a useful label for both endogenous and antibody-conjugated AP activity as detected by flow cytometry. Its spectral characteristics allow it to be combined with a variety of fluorochromes for multiparametric analysis. Cytometry 43:117-125, 2001. Published 2001 Wiley-Liss, Inc.     

Cryptomonad algal phycobiliproteins as fluorochromes for extracellular and intracellular antigen detection by flow cytometry

BACKGROUND: Phycobiliproteins play an important role in fluorescent labeling, particularly for flow cytometry. The spectral properties of R-phycoerythrin (R-PE) and allophycocyanin (APC) have made them the dominant reagents in this class of fluorochromes. In this study, we evaluate a lesser-known but potentially important series of low-molecular weight cryptomonad-derived phycobiliproteins (commercially termed the CryptoFluortrade mark dyes) for their applicability to flow cytometry, both in extracellular and intracellular labeling applications. METHODS: Several cell lines were labeled with biotin-conjugated antibodies against expressed extracellular surface proteins, followed by streptavidin conjugates of three cryptomonad phycobiliproteins (CryptoFluor-2, CryptoFluor-4, and CryptoFluor-5). Cells were then analyzed by flow cytometry using a variety of laser lines and emission filters to establish the optimal excitation/emission characteristics for each fluorochrome. Some cells were permeabilized and labeled for intracellular antigens, also using the cryptomonad fluorochromes. Where appropriate, parallel samples were labeled with other fluorochromes (including R-PE, APC, the cyanin dyes Cy3 and Cy5, and others) to gauge the performance of the cryptomonad fluorochromes against fluorescent labels previously evaluated for flow cytometry. RESULTS: CryptoFluor-2 possessed excitation/emission maxima similar to those of APC and Cy5, with good excitation in the red (HeNe laser 632 nm) and strong emission in the far red (660 nm). CryptoFluor-4 possessed excitation/emission maxima similar to those of Cy3, with optimal excitation in the green (Kr 530 nm) and strong emission in the yellow/orange (585 nm). CryptoFluor-5 possessed excitation/emission maxima similar to those of lissamine rhodamine, with optimal excitation in the yellow (Kr 568 nm) and emission in the orange (610 nm). All cryptomonad fluorochromes gave satisfactory results for both intracellular and extracellular labeling, with detection sensitivities that were comparable or better than traditional phycobiliproteins and low- molecular weight synthetic fluorochromes such as the cyanin dyes. CONCLUSIONS: The CryptoFluor fluorochromes were applicable to flow cytometric immunodetection, with excitation and emission conditions commonly found on multilaser instruments. Performance of several of these dyes was at least comparable to existing fluorescent labels. The low molecular weights (30-60 kd) of phycobiliproteins may make them particularly useful in intracellular antigen detection. Cytometry 44:16-23, 2001. Published 2001 Wiley-Liss, Inc.     

A unified procedure for conservative (morphology) and integral (DNA and immunophenotype) cell staining for flow cytometry

BACKGROUND: Current methods for multiparameter DNA flow cytometry suffer from several limitations. These include significant modifications of cell morphological parameters, the impossibility to counterstain cells with certain fluorochromes, and laborious tuning of the instrument that, for some procedures, must be equipped with an ultraviolet (UV) laser. To overcome these problems, we developed a novel method for the simultaneous analysis of morphological parameters, four-color immunophenotyping, and stoichiometric DNA labeling using a bench-top flow cytometer. METHODS: The method consists of a mild permeabilization/fixation treatment at room temperature, followed by labeling with fluorochrome-conjugated monoclonal antibodies (mAbs) and with the DNA dye 7-aminoactinomycin D (7-AAD) at 56 degrees C. RESULTS: Using this method, we analyzed resting peripheral blood mononucleated cells (PBMC), proliferating T cells cultured in the presence of interleukin-2 (IL-2), and lymphoblastoid B cells. Lymphocytes, monocytes, and lymphoblasts treated by this procedure retained differential light scattering (DLS) characteristics virtually identical to those of untreated cells. This allowed regions to be drawn on forward scatter (FSC) and side scatter (SSC) cytograms resolving different cell populations. DLS were preserved well enough to distinguish large lymphoblasts in the S or G2/M phases from small G0/G1 cells. Also, stainability with fluorescein-isothiocyanate (FITC), R-phycoerythrin (PE), allophycocyanin (APC)-conjugated mAbs was generally preserved. DNA labeling with 7-AAD was of quality good enough to permit accurate cell cycle analysis. CONCLUSIONS: The method described here, which we called integral hot staining (IHS), represents a very simple, reproducible, and conservative assay for multiparameter DNA analysis using a bench-top flow cytometer. Last but not least, the cytometer tuning for multiparameter acquisition is straightforward.     

Hapten-mediated immunopurification of membrane proteins labeled with fluorescein derivatives

The behavior of cell surface components labeled with fluorochromes can be studied by fluorescence microscopy and spectroscopy; further structural analyses would be facilitated by purification of the labeled components. We have developed a protocol for identifying the targets for labeling with fluorescein derivatives, by using 125I- diiodofluorescein isothiocyanate ( 125IFC ) and for isolating the labeled components with anti-IFC immunoadsorbents. Anti-IFC antibodies obtained from rabbits immunized with IFC-hemocyanin were purified by affinity chromatography and coupled to CNBr-activated Sepharose 4B. The anti-IFC immunoadsorbents could then be used to isolate the entire set of 125IFC -proteins from crude detergent extracts of labeled sea urchin sperm, with a 70% yield and a purification of more than 250 fold. Nonspecific binding of unlabeled proteins to the immunoadsorbent was insignificant. When the immunoadsorbent IFC-protein complex was used directly as an immunogen, antibodies were obtained that reacted with the underivatized proteins that were targets for IFC labeling, as indicated by immunoblotting after gel electrophoresis. The antibodies also reacted with the surface of unlabeled sperm as shown by immunofluorescence. Thus, by treating the IFC-sperm proteins as a class, we obtained antibodies that recognized the unlabeled proteins in situ or in cell extracts. This approach should be generally useful in obtaining reagents directed against specific cell surface components.     

Caffeine dissociates complexes between DNA and intercalating dyes: application for bleaching fluorochrome-stained cells for their subsequent restaining and analysis by laser scanning cytometry

BACKGROUND: Removal of the nucleic acid-bound fluorochrome is desirable when stained cells have to be reanalyzed using other fluorochromes. It is also often desirable to remove DNA-bound antitumor drugs from drug-treated cells, to improve cell staining. We have previously observed that in aqueous solutions, the methylxanthine caffeine (CFN) decreases interactions between planar aromatic molecules such as intercalating dyes or antitumor drugs and nucleic acids. The aim of this study was to explore whether this property of CFN can be utilized to remove the DNA-bound intercalating dyes propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) from the cells and whether the bleached cells can be restained and reanalyzed. METHODS: HL-60 cells were fixed in 70% ethanol and their DNA was stained with PI or 7-AAD. The cells were then rinsed with a 0.05 M solution of CFN in phosphate-buffered saline (PBS) or with PBS alone. The decrease in intensity of cell fluorescence during rinsing was measured by laser scanning cytometry (LSC) to obtain the bleaching kinetics of individual cells. The bleached cells were then restained with PI, 7-AAD, or the protein-specific fluorochrome sulforhodamine 101(S101). Their fluorescence was measured again by LSC. In addition, free DNA was subjected to gel electrophoresis, DNA bands in the gels were stained with ethidium bromide (EB), and the gels were rinsed with a solution of CFN or PBS to bleach the DNA band's fluorescence. RESULTS: Rinsing the PI or 7-AAD-stained cells with solutions of CFN led to nearly complete removal of PI and a more than 75% decrease in 7-AAD fluorescence after 10 min. The rinse with PBS decreased the PI cell fluorescence intensity by less than 30% and the 7-AAD fluorescence by about 50%. The differences in kinetics of PI or 7-AAD removal by CFN from G2/M versus G1 cells suggest that these intercalators bind more strongly to DNA in chromatin of G2/M than G1 cells. The CFN-bleached cells were then successfully stained with S101 and again with PI or 7-AAD. The bivariate analysis of the LSC merged files of the cells sequentially stained with PI and S101 revealed typical DNA/protein distributions. The fluorescence of EB-stained DNA bands in gels was also nearly completely removed by rinsing gels in 0.05 M CFN; PBS alone had a distinctly lesser effect. CONCLUSION: Solutions of CFN can dissociate the DNA-bound PI, 7-AAD, EB, and possibly other intercalating fluorochromes. The bleached cells can be restained and reanalyzed by LSC. This approach can also be used to remove such fluorochromes from nucleic acids immobilized in gels and perhaps in other solid matrices. Analysis of the kinetics of fluorochrome removal from cells can possibly be used to study their binding affinities to nucleic acids in situ.     

Complete assignment of the methionyl carbonyl carbon resonances in switch variant anti-dansyl antibodies labeled with [1-13C]methionine

A 13C NMR study is reported of switch variant anti-dansyl antibodies developed by Dangl et al. [(1982) Cytometry 2, 395-401], who had used the fluorescence-activated cell sorter to select and clone these variants. These switch variant antibodies possess the identical VH, VL, and CL domains in conjunction with different heavy chain constant regions. In the present study, switch variant antibodies of IgG1, IgG2a, and IgG2b subclasses were used along with a short-chain IgG2a antibody, in which the entire CH1 domain is deleted. The switch variant antibodies were specifically labeled with [1-13C]methionine by growing hybridoma cells in serum-free medium. Assignments of all the methionyl carbonyl carbon resonances have been completed by using the intact antibodies along with their fragments and recombined proteins in which either heavy or light chain is labeled. A double labeling method [Kainosho, M., & Tsuji, T. (1982) Biochemistry 21, 6273-6279] has played a crucial role in the process of the spectral assignments. The strategy used for the assignments has been described in detail. In incorporating 15N-labeled amino acids into the antibodies for the double labeling, isotope dilution caused a serious problem except in the cases of [alpha-15N]lysine and [15N]threonine, both of which cannot become the substrate of transaminases. It was found that beta-chloro-L-alanine is most effective in suppressing the isotope scrambling. So far, spectral assignments by the double labeling method have been possible with 15N-labeled Ala, His, Ile, Lys, Met, Ser, Thr, Tyr, and Val.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1) selection of fluorescent dyes having minimal spectral overlap is complicated, and 2) quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH³ resin composites. Specimens were subsequently immersed for 60 sec in either Biotène PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; α=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p<0.05) on both resin composites, whereas LTO did not produce differences (p>0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.     

Simultaneous cell cycle analysis and two-color surface immunofluorescence using 7-amino-actinomycin D and single laser excitation: applications to study of cell activation and the cell cycle of murine Ly-1 B cells

The DNA-binding, fluorescent dye 7-amino-actinomycin D (7AAD) is efficiently excited by the 488 nm laser line commonly used in flow cytometry, but yields fluorescence emission further into the red spectrum than alternative DNA-specific fluorochromes. In this report, we show that the spectral properties of 7AAD allow single-laser analysis of DNA content and cell cycle simultaneously with two cell surface markers labeled with fluorescein (green)-and phycoerythrin (orange)-conjugated antibodies. The use of 7AAD makes three-color analysis practical and feasible, using the most widely available flow cytometric instruments. The power of this technique was demonstrated in two systems. Staining of human peripheral blood lymphocytes (PBL) with 7AAD was demonstrated to be dependent on cell activation and chromatin conformation; PHA-stimulated cells which have become activated and express IL 2 receptors had greater 7AAD fluorescence than nonactivated, IL 2 receptor-negative cells. Cell cycle analysis of mouse splenocytes stained with fluorescent antibodies to IgM and to Ly-1 demonstrated that the proportion of S and G2 phase cells in native spleen varies strongly among the subsets of cells identified with these markers. Of particular interest was the striking finding that the Ly-1+/IgM+ subset (Ly-1 B cells) is greatly enriched for cells in the S phase fraction. This is important because Ly-1 B cells have been associated with the production of autoantibodies, and is consistent with reports that these cells have a lymphoblastoid or a plasmablast morphology. We hypothesize that Ly-1 B cells may belong to a subset of in vivo activated cells which are either rapidly proliferating or are arrested in S phase.     

Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester peptide chloromethyl ketones. II. Properties of thrombin derivatives as reporters of prothrombin fragment 2 binding and specificity of the labeling approach for other proteinases.

The behavior of an array of fluorescent human alpha-thrombin derivatives in reporting binding of the fragment 2 domain of prothrombin was characterized as a representative application of the active-site-selective labeling approach to studies of blood coagulation proteinase regulatory interactions. An array of 16 thrombin derivatives was prepared by affinity labeling of the proteinase active site with the thioester peptide chloromethyl ketones, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl or N alpha-[(acetylthio)acetyl]-D-Phe-Phe-Arg-CH2Cl, followed by selective modification of the NH2OH-generated thiol group on the covalently incorporated inhibitors with each of eight thiol-reactive fluorescence probes. The changes in probe fluorescence intensity of the derivatives, signaling changes in the environment of the catalytic site associated with fragment 2 binding, appeared to be a unique and unpredictable function of the structure of the probe and the connecting peptide. These results demonstrated the utility of the labeling approach for overcoming the problem of not being able to predict which fluorescent label will provide the most useful proteinase derivative for investigating an interaction by enabling a greater variety of them to be prepared and screened for those with the most desirable properties. To determine whether the approach could be extended to other proteinases, the specificity of labeling with the fluorescence probe iodoacetamide, 5-(iodoacetamido)fluorescein, by use of the two thioester inhibitors was evaluated for several other blood coagulation proteinases and related trypsin-like enzymes. All of the proteinases were labeled in an active-site-selective manner. The combined results of quantitating the labeling reactions for the proteinase and inhibitor combinations studied thus far showed active-site-specific incorporation of 0.98 +/- 0.10 mol of inhibitor/mol of active sites and 0.92 +/- 0.11 mol of probe/mol of active sites, representing an overall greater than or equal to 93% site-specificity of labeling. These results demonstrated the broad applicability of the labeling approach for fluorescence studies of proteinases that differ greatly in their catalytic specificities.     

Interactions of fluorescent mercurial compounds and acidic fluorochromes with isolated living cells and nuclei

Summary Two fluorescent mercurials (fluorescein mercuric acetate and merbromin) and two acidic fluorochromes (brilliant sulfoflavine and primuline) were tested as supravital fluorochromes and compared with the fluorescent probe for hydrophobic groups, 8-anilino-1-naphthalene-sulfonic acid (ANS). Neither the mercurials nor the acidic fluorochromes appeared to penetrate intact cells, but all of the dyes fluorochromed damaged cells in a characteristic fashion. Expriments were then undertaken on nuclei isolated in 0.25 M sucrose. The fluorescent mercurials produced fluorescence of the nuclear envelope and nucleoli. More generalized fluorescence was induced if nuclei remained for prolonged periods in saline solutions balanced for intact cells or in nuclei exposed to 0.2 N hydrochloric acid. Acidic fluorochromes produced a more generalized distributional pattern of fluorescence. Primuline produced substantially more intense nuclear fluorescence than brilliant sulfoflavine at equimolar concentrations. Considered as a whole, these results indicate that an examination of the interaction of fluorescent dyes with unfixed cellular components could prove to be a useful tool in cell biology, particularly in the investigation of nuclear function.Supported in part by GRS-FR-5394 to the Albany Medical College.     

Nitrogen-15 labeling effectiveness of two tropical legumes

Nitrogen-15 foliar applications for the production of field-labeled plant tissues may achieve more effective labeling of plant shoot and root tissues and minimize directly labeling the soil N fraction as occurs when¹⁵ N is soil applied. Consequently, foliar-labeled plant tissues should be better suited for subsequent ¹⁵N mineralization studies. A field experiment was conducted to determine the effectiveness of ¹⁵N-labeling and the accumulation of ¹⁵N in various plant parts of two tropical legumes. Desmodium ovalifolium Guillemin and Perrottet and Pueraria phaseoloides (Roxb.) Benth., grown in 0.5 m² microplots, were labeled with foliar-applied urea containing 99 atom% ¹⁵N. Plants in each microplot received a total of 0.1698 g ¹⁵N that was applied all at once or split equally into two, three or four applications. Legume shoots and roots and soil were destructively harvested and analyzed for total ¹⁵N content. Averaged over both legumes and foliar application rates, total plant (shoots, flowers, leaf litter, and roots) recovery was approximately 79% of the ¹⁵N applied. The soil contained 3% of the ¹⁵N applied, of which 2.5 and 0.5% were in the inorganic and organic fractions, respectively. Nitrogen-15 recovery in shoots (76%) was sixty-five fold greater than in roots (1%) and about nineteen fold greater than the sum of roots and soil (4.1%), a much greater percent recovery than observed in other foliar labeling studies. Averaged over all four foliar split-application rates, ¹⁵N recovery by Desmodium shoots was greater than Pueraria. Results demonstrate that ¹⁵N foliar application to legumes is an effective method for labeling, resulting in atom% excess ¹⁵N levels and ¹⁵N recoveries comparable to those reported with the more traditional soil-labeling approach. Another advantage of this method is a nondestructive, in situ labeling method that permits separation of shoot and root residual N contribution to subsequent crops in N tracer studies.     

The purine nucleotide cycle and ammoniagenesis in rat kidney tubules

The contribution of the purine nucleotide cycle to renal ammoniagenesis was examined in cortical tubule suspensions prepared from acidotic rats and incubated with [alpha-15N]glutamine, [15N]glutamate, or [15N]aspartate. Labeling of ammonia and adenine nucleotides was determined after enzymatic transformations designed to circumvent the technical problem that 15NH3 and H2O have the same nominal mass. Labeling of the adenine nucleotide was undetectable (less than 10%) even after 1 h of incubation. From the measured concentrations of adenine nucleotides and ammonia and the labeling of the ammonia, the flux through the purine nucleotide cycle was calculated to account for less than 1% of the deamination of alpha-amino groups from all three substrates. The glutamate dehydrogenase reaction is therefore the likely pathway for deamination. The rate of 15NH3 production from [alpha-15N]glutamine was two or three times greater than from added [15N]glutamate, indicating a preference for intracellularly generated glutamate. 15NH3 production from added [15N]aspartate was similar to and perhaps slightly greater than that from added [15N]glutamate.     

Correlated analysis of cellular DNA, membrane antigens and light scatter of human lymphoid cells

Flow cytometric correlated analysis of membrane antigens, DNA, and light scatter was performed on human lymphoid cells using fluorescein (FITC)-conjugated antibodies to label B- and T-cell antigens and propidium iodide (PI) to stain DNA after ethanol fixation and RNase treatment. A FACS II flow cytometer was modified to obtain digitized measurements of two color fluorescence and light scatter emissions, simultaneously. Software was written to allow single parameter analysis or correlated analysis of any two of the three parameters acquired. Ethanol fixation preserved FITC surface labeling for at least 15 weeks, but produced marked changes in light scatter. No changes in FITC distributions were observed after RNase treatment and PI staining, and the presence of FITC labeling did not affect DNA distributions. Within heterogeneous cell populations, the DNA distribution of cell subpopulations identified by a membrane antigen was clearly demonstrated.     

Fluorescence-intensity multiplexing: simultaneous seven-marker, two-color immunophenotyping using flow cytometry.

BACKGROUND: Conventional immuno-based multiparameter flow cytometric analysis has been limited by the requirement of a dedicated detection channel for each antibody-fluorophore set. To address the need to resolve multiple biological targets simultaneously, flow cytometers with as many as 10-15 detection channels have been developed. In this study, a new Zenon immunolabeling technology is developed that allows for multiple antigen detection per detection channel using a single fluorophore, through a unique method of fluorescence-intensity multiplexing. By varying the Zenon labeling reagent-to-antibody molar ratio, the fluorescence intensity of the antibody-labeled cellular targets can be used as a unique identifier. Although demonstrated in the present study with lymphocyte immunophenotyping, this approach is broadly applicable for any immuno-based multiplexed flow cytomety assay. METHODS: Lymphocyte immunophenotyping of 38 clinical blood specimens using CD3, CD4, CD8, CD16, CD56, CD19, and CD20 antibodies was performed using conventional flow cytometric analysis and fluorescence-intensity multiplexing analysis. Conventional analysis measures a single antibody-fluorophore per photomultiplier tube (PMT). Fluorescence-intensity multiplex analysis simultaneously measures seven markers with two PMTs, using Zenon labeling reagent-antibody complexes in a single tube: CD19, CD4, CD8, and CD16 antibodies labeled with Zenon Alexa Fluor 488 Mouse IgG(1) labeling reagent and CD56, CD3, and CD20 antibodies labeled with Zenon R-Phycoerythrin (R-PE) Mouse IgG(1) or IgG(2b) labeling reagents. RESULTS: The lymphocyte immunophenotyping results from fluorescence-intensity multiplexing using Zenon labeling reagents in a single tube were comparable to results from conventional flow cytometric analysis. CONCLUSIONS: Simultaneous evaluation of multiple antigens using a single fluorophore can be performed using antibodies labeled with varying ratios of a Zenon labeling reagent. Labeling two sets of antibodies with different Zenon labeling reagents can generate characteristic and distinguishable multivariate patterns. Combining multiple antibodies and fluorescent labels with fluorescence intensity multiplexing enables the resolution of more cellular targets than detection-channels, allowing sophisticated multiparameter flow cytometric studies to be performed on less complex 2- or 3-detection-channel flow cytometers. For typical biological samples, approximately 2-4 cellular targets per detection channel can be resolved using this technique.