texas-red and 4-4-difluoro-4-bora-3a-4a-diaza-s-indacene

Fluorescence measurements of the sterol analog 23-(dipyrrometheneboron difluoride)-24-norcholesterol (BODIPY-cholesterol) are used to compare the effects of cholesterol (Chol) in monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Chol and chicken egg sphingomyelin (SM)/DOPC/Chol. Monolayers are formed using the Langmuir-Blodgett technique and compared at surface pressures of 8 and 30 mN/m. In particular, these ternary lipid mixtures are compared using both ensemble and single-molecule fluorescence measurements of BODIPY-cholesterol. In mixed monolayers incorporating 0.10 mol % BODIPY-cholesterol, fluorescence microscopy measurements as a function of cholesterol added reveal similar trends in monolayer phase structure for both DPPC/DOPC/Chol and SM/DOPC/Chol films. With a probe concentration reduced to ∼10(-8) mol % BODIPY-cholesterol, single-molecule fluorescence measurements using defocused polarized total internal reflection microscopy are used to characterize the orientations of BODIPY-cholesterol in the monolayers. Population histograms of the BODIPY emission dipole tilt angle away from the membrane normal reveal distinct insertion geometries with a preferred angle observed near 78°. The measured angles and populations are relatively insensitive to added cholesterol and changes in surface pressure for monolayers of SM/DOPC/Chol. For monolayers of DPPC/DOPC/Chol, however, the single-molecule measurements reveal significant changes in the BODIPY-cholesterol insertion geometry when the surface pressure is increased to 30 mN/m. These changes are discussed in terms of a squeeze-out mechanism for BODIPY-cholesterol in these monolayers and provide insight into the partitioning and arrangement of BODIPY-cholesterol in ternary lipid mixtures.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Boron Compounds; Chickens; Cholesterol; Fluorescent Dyes; Microscopy, Fluorescence; Microscopy, Polarization; Phosphatidylcholines; Phosphatidylethanolamines; Sphingomyelins; Xanthenes

Topics: Boron Compounds; Cholesterol; Electrophoresis; Fluorescent Dyes; Lipid Bilayers; Membranes, Artificial; Staining and Labeling; Xanthenes

The uptake of different surfactant lipids-dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol (PG), or phosphatidylinositol (PI)-and liposomes with a surfactant-like composition by alveolar type II cells (alveolar type II cells) and macrophages (alveolar macrophages) was studied in vitro. Fluorescent-labeled liposomes containing either 86% of the studied lipid, i.e., DPPC, PG, PI, and 6% labeled phosphatidylethanolamine (PE) and 8% cholesterol or a lipid mixture similar to surfactant (DPPC, PG, PI, phosphatidylcholine, PE, and cholesterol in a weight ratio of 55:8:2:21:8:6) were incubated with alveolar macrophages and alveolar type II cells. The cell-associated fluorescence assessed by flow cytometry demonstrated a higher uptake of PG and PI by both alveolar macrophages and alveolar type II cells, and a lower uptake of DPPC by alveolar macrophages. In addition, fewer alveolar type II cells take up DPPC, whereas there are no differences for the alveolar macrophages in the number of cells involved in the uptake. Competition experiments with Texas Red-labeled liposomes and either DPPC liposomes or PI liposomes labeled with Bodipy indicated that all these liposomes are internalized via the same pathway by alveolar cells. Thus, lipid composition directly influences the (re)uptake of surfactant.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Biological Transport; Boron Compounds; Cells, Cultured; Fluorescent Dyes; Liposomes; Macrophages, Alveolar; Male; Phosphatidylglycerols; Phosphatidylinositols; Pulmonary Alveoli; Pulmonary Surfactants; Rats; Rats, Sprague-Dawley; Xanthenes

A functional fluorescent neurokinin NK2 receptor, EGFP-NK2, was previously used to follow, by fluorescence resonance energy transfer measurements in living cells, the binding of its fluorescently labeled agonist, bodipy-neurokinin A (NKA). Local agonist application suggested that the activation and desensitization of the NK2 receptors were compartmentalized at the level of the plasma membrane. In this study, fluorescence recovery after photobleaching experiments are carried out at variable observation radius (vrFRAP) to probe EGFP-NK2 receptor mobility and confinement. Experiments are carried out at 20 degrees C to maintain the number of receptors constant at the cell surface during recordings. In the absence of agonist, 35% EGFP-NK2 receptors diffuse within domains of 420 +/- 80 nm in radius with the remaining 65% of receptors able to diffuse with a long range lateral diffusion coefficient between the domains. When cells are incubated with a saturating concentration of NKA, 30% EGFP-NK2 receptors become immobilized in small domains characterized by a radius equal to 170 +/- 50 nm. Biochemical experiments show that the confinement of EGFP-NK2 receptor is not due to its association with rafts at any given time. Colocalization of the receptor with beta-arrestin and transferrin supports that the small domains, containing 30% of activated EGFP-NK2, correspond to clathrin-coated pre-pits. The similar amount of confined EGFP-NK2 receptors found before and after activation (30-35%) is discussed in term of putative transient interactions of the receptors with preexisting scaffolds of signaling molecules.

Topics: Arrestins; beta-Arrestins; Boron Compounds; Cell Line; Cell Membrane; Clathrin; Diffusion; Dose-Response Relationship, Drug; Fluorescence Recovery After Photobleaching; Fluorescent Dyes; Genetic Vectors; Green Fluorescent Proteins; Humans; Immunoblotting; Lipids; Microscopy, Confocal; Microscopy, Fluorescence; Models, Chemical; Neurokinin A; Octoxynol; Protein Structure, Tertiary; Receptors, Neurokinin-2; Temperature; Time Factors; Transfection; Transferrin; Xanthenes

Eosinophils and their products play a major role in inflammatory reactions associated with asthma and allergic diseases. There is a growing body of evidence that eosinophils synthesize, store, and release bioactive cytokines and chemokines with the potential to contribute to local inflammatory changes. Fluorescein isothiocyanate (FITC) has been widely used as an immunofluorescent conjugate for antibodies specific for detection of these molecules. However, FITC is an ionic fluorochrome (negatively charged) which binds strongly to positively charged eosinophil granule proteins. We developed new methods to prevent charge-based interactions of ionic fluorochromes with granule proteins, and optimised immunofluorescent staining techniques for eosinophils. An antibody to interleukin-6 (IL-6) was used to optimise this procedure for eosinophil-derived granule proteins. We attempted to block nonspecific binding of FITC-labelled anti-IL-6 using normal human IgG, foetal calf serum (FCS), bovine serum albumin (BSA), and goat, horse, and normal human sera at concentrations ranging between 1-10%. Only human IgG (2%; 20 mg/ml) was able to reduce background fluorescence. These results were confirmed using Texas Red conjugates. We also used antibodies conjugated to a nonionic fluorochrome, BODIPY FL, to detect IL-6 in eosinophils. Unlike FITC, BODIPY FL-conjugated antibodies did not require strong blocking conditions (2% BSA). We recommend that a neutral fluorochrome (BODIPY FL) should be used for immunofluorescence studies in eosinophils. Alternatively, strong blocking conditions may be used to decrease background binding of FITC-conjugated antibodies.

Topics: Animals; Antigen-Antibody Reactions; Artifacts; Asthma; Blood Proteins; Boron Compounds; Cattle; Cytoplasmic Granules; Eosinophil Granule Proteins; Eosinophils; False Positive Reactions; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Goats; Horses; Humans; Immunoglobulin G; Interleukin-6; Microscopy, Confocal; Protein Binding; Ribonucleases; Serum Albumin, Bovine; Xanthenes

We have prepared casein conjugates of two BODIPY dyes for use as fluorogenic protease substrates in homogeneous assays. Both conjugates are labeled to such an extent that the dyes are efficiently quenched in the protein, yielding virtually nonfluorescent substrate molecules. These fluorogenic substrates release highly fluorescent BODIPY dye-labeled peptides upon protease digestion, with fluorescence increases proportional to enzyme activity. These quenched substrates are suitable for the continuous assay of enzymatic activity using standard fluorometers, filter fluorometers, or fluorescence microplate readers using either fluorescein excitation and emission wavelengths to measure BODIPY FL casein hydrolysis or Texas Red wavelengths to detect proteolysis of BODIPY TR-X casein. Most current techniques for detecting protease activity, such as the fluorescein thiocarbamoyl casein (FTC-casein) protease assay, require extensive manipulation, including separation steps, and are therefore labor intensive and error-prone. In comparison, we found the BODIPY dye-labeled casein protease assays to be simple and precise and to have greater sensitivity and a broader dynamic range of detection than the FTC-casein assay. We were able to sensitively detect the activities of a wide variety of enzymes with these new substrates, including serine, acid, sulfhydryl, and metalloproteases. We also found the assay suitable for quantitating protease inhibitor concentrations and for real-time analysis of proteolysis.

Topics: Animals; Boron Compounds; Caseins; Endopeptidases; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique, Direct; Fluorescent Dyes; Humans; Hydrolysis; Protease Inhibitors; Spectrometry, Fluorescence; Xanthenes

The performance characteristics of two sets of triple-band epifluorescence filters have been evaluated for use with digitally enhanced fluorescence microscopy. Use of such filters, at most, requires movement of the excitation filter, while the dichroic and emission filters remain fixed, allowing multi-wavelength imaging to be performed on standard microscopes. The dyes appropriate for use with these particular filters include Texas Red (TR), Bodipy (BD), FITC and Cascade Blue (CB), four fluorophores now commonly conjugated to both immunochemical probes and other proteins and lipids of biological interest. Good spectral separation existed for most experimental conditions allowing accurate localization of the different fluorophores during multi-wavelength imaging. Anomalous responses were observed during near-UV excitation at high concentration for some dyes. Scanning spectrofluorometry demonstrated that concentration-dependent spectral shifts occurred, resulting in large increases in near-UV absorbance. Despite the complexity of concentration and dye-interaction effects, quantitative measurements of dye concentration could be made, even in regions of multiple dye co-localization. Therefore, multi-band pass filters are an additional valuable approach for performing quantitative fluorescence microscopic imaging.

Topics: Animals; Boron Compounds; Evaluation Studies as Topic; Filtration; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Mice; Microscopy, Fluorescence; Organometallic Compounds; Organophosphorus Compounds; Serum Albumin, Bovine; Spectrometry, Fluorescence; Xanthenes