Fluorescence polarization of six membrane probes in embryonal carcinoma cells after differentiation as measured on a FACS II cell sorter

Fluorescence polarization measurements on a FACS II cell sorter were compared with static measurements on a spectrofluorimeter using calibration solutions and Hoechst 33258-labeled cells. For the flow cytometric measurements on the FACS we used a pseudodepolarizer for normalization of the output of the two photomultipliers. The results showed that fluorescein and fluoresceinated bovine serum albumin (BSA) solutions gave identical values on both instruments. The mean value for fluorescence polarization of Hoechst 33258-labeled cells as measured on the FACS was the same as the value obtained with the spectrofluorimeter. Subsequently the fluorescence polarization of six different membrane probes was determined using differentiating embryonal carcinoma cells as a model system. Differentiation was induced by treatment of the cells with retinoic acid together with cyclic AMP. With diphenylhexatriene (DPH) the fluorescence polarization increased from I/I = 1.55 to 1.74 upon differentiation. With a charged analog of DPH (TMA-DPH) fluorescence polarization increased from I/I = 1.87 to 2.02. No appreciable changes in fluorescence polarization were observed in this cell system when anthroyloxysterate probes (12-AS, 9-AS, 6-AS, 2-AS) were used.     

Simultaneous detection of several oligonucleotides by time-resolved fluorometry: the use of a mixture of categorized microparticles in a sandwich type mixed-phase hybridization assay.

Porous, uniformly sized (50 micrometer) glycidyl methacrylate/ethylene dimethacrylate particles (SINTEF) were used as a solid phase to construct a sandwich type hybridization assay that allowed simultaneous detection of up to six oligonucleotides from a single sample. The assay was based on categorization of the particles by two organic prompt fluorophores, viz. fluorescein and dansyl, and quantification of the oligonucleotide hybridization by time-resolved fluorometry. Accordingly, allele-specific oligodeoxyribonucleotide probes were assembled on the particles by conventional phosphoramidite strategy using a non-cleavable linker, and the category defining fluorescein and/or dansyl tagged building blocks were inserted in the 3'-terminal sequence. An oligonucleotide bearing a photoluminescent europium(III) chelate was hybridized to the complementary 3'-terminal sequence of the target oligonucleotide, and the resulting duplex was further hybridized to the particle-bound allele-specific probes via the 5'-terminal sequence of the target. After hybridization each individual particle was subjected to three different fluorescence intensity measurements. The intensity of the prompt fluorescence signals of fluorescein and dansyl defined the particle category, while the europium(III) chelate emission quantified the hybridization. The length of the complementary region between the target oligonucleotide and the particle-bound probe was optimized to achieve maximal selectivity. Furthermore, the kinetics of hybridization and the effect of the concentration of the target oligomer on the efficiency of hybridization were evaluated. By this approach the possible presence of a three base deletion (DeltaF508), point mutation (G542X) and point deletion (1078delT) related to cystic fibrosis could unequivocally be detected from a single sample.     

Fluorogenic approach to evaluating prodrug hydrolysis and stability in live cells

Fluorescein diester which is conjugated with cell membrane permeable Arg9 peptide was proposed as probe for ester prodrug stability and drug release study in living cells. α-Amino protected d-Val and l-Ala which bear differently hindered side chains were used to afford model diesters of 5-maleimide-fluorescein. Such fluorescein diesters were further conjugated with a Cys containing cell membrane permeable Arg9 peptide via thiol-ene crosslink reaction. The resulted conjugates of fluorescein diester and Arg9 peptide were purified with HPLC and characterized with MALDI-TOF MS. Upon incubation with cultured cells, the fluorescein diesters were delivered into the cells, the following hydrolysis of fluorescein diesters and release of fluorescein inside living cells were observed by monitoring the fluorescence accumulation. Fluorescence microscopic imaging studies of HeLa cells treated with fluorescein l-Ala diester show strong fluorescence accumulation in 30?min indicating fast hydrolysis of fluorescein diester and fluorescein release; in contrast d-Val diester remains stable inside cells evidenced by margin fluorescence formation. Further flowcytometry studies on the fluorescein diester-Arg9 conjugate treated cells show that the hydrolysis t_1/2 for l-Ala diester is 15?min. The results also show that Arg9 peptide not only transports the ester probes into cell efficiently but also can retain and concentrate hydrolytic product fluorescein inside cells so that the accumulated fluorescence can be accurately quantified. This fluorogenic probe approach provides feasible applications in dynamic studies on ester prodrug hydrolysis and release, facilitating screening and optimization of prodrug structures in living cell settings.     

Synthesis of chlorinated fluoresceins for labeling proteins

Two novel chlorinated fluoresceins 4,7,2',7'-tetrachloro-6-(5-carboxypentyl)fluorescein (8a) and 4,7,4',5'-tetra-chloro-6-(5-carboxypentyl)fluorescein (8b) were synthesized as fluorescent probes for labeling proteins. These two fluoresceins contain 6-aminohexanoic acid as spacer linker to minimize the fluorescence quenching of the fluorescein molecules by the proteins to be labeled.     

Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization

DNA was assayed in a homogeneous format using DNA probes containing hybridization-sensitive labels. The DNA probes were prepared from complementary DNA strands in which one strand was covalently labeled on the 5'-terminus with fluorescein and the complementary strand was covalently labeled on the 3'-terminus with a quencher of fluorescein emission, either pyrenebutyrate or sulforhodamine 101. Probes prepared in this manner were able to detect unlabeled target DNA by competitive hybridization producing fluorescence signals which increased with increasing target DNA concentration. A single pair of complementary probes detected target DNA at a concentration of approximately 0.1 nM in 10 min or about 10 pM in 20-30 min. Detection of a 4 pM concentration of target DNA was demonstrated in 6 h using multiple probe pairs. The major limiting factors were background fluorescence and hybridization rates. Continuous monitoring of fluorescence during competitive hybridization allowed correction for variable sample backgrounds at probe concentrations down to 20 pM; however, the time required for complete hybridization increased to greater than 1 h at probe concentrations below 0.1 nM. A promising application for this technology is the rapid detection of amplified polynucleotides. Detection of 96,000 target DNA molecules in a 50-microliters sample was demonstrated following in vitro amplification using the polymerase chain reaction technique.     

Quantitative imaging and statistical analysis of fluorescence in situ hybridization (FISH) of Aureobasidium pullulans

Image and multifactorial statistical analyses were used to evaluate the intensity of fluorescence signal from cells of three strains of A. pullulans and one strain of Rhodosporidium toruloides, as an outgroup, hybridized with either a universal or an A. pullulans 18S rRNA oligonucleotide probe in direct or indirect FISH reactions. In general, type of fixation (paraformaldehyde or methanol-acetic acid) had no apparent effect on cell integrity and minimal impact on fluorescence. Permeabilization by enzyme treatment for various times, though needed to admit high Mw detection reagents (avidin-FITC) in indirect FISH, tended to nonspecifically degrade cells and lower the signal. Digestion was unnecessary and undesirable for the directly labelled probes. Multilabelled (five fluorescein molecules) probes enhanced fluorescence about fourfold over unilabelled probes. Overall, direct FISH was preferable to indirect FISH and is recommended especially for studies of microbes on natural substrata.     

Towards a Non-Terminal Viability Assay for Foraminiferan Protists

ABSTRACT. Epifluorescence microscopy and spectrofluorimetry were investigated as possible non-terminal methods to distinguish live from dead foraminifera. Seven fluorogenic probes (diacetates of fluorescein [FDA], carboxyfluorescein, dichlorofluorescein, and carboxyeosin; AM-esters of biscarboxyethylcarboxyfluorescein [BCECF-AM], calcein, and calcein blue) were tested on Allogromia laticollaris . The probes that consistently produced the brightest fluorescence signals (BCECF-AM and FDA) were judged non-toxic to Allogromia , on the basis of short-term pseudopodial deployment and long-term reproduction assays. Once protocols were established, these two probes were tested on 13 additional benthic foraminiferal species. We found that BCECF-AM is the most suitable probe for direct epifluorescence microscopy of metabolically active foraminifera, especially tectinous and transparent calcareous species. Using spectrofluorimetry, FDA showed promise for opaque species because fluorescence is detected in the incubation media after its release from the cell. However, both approaches could only be used with confidence in light of appropriate controls established for each species examined.     

Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester peptide chloromethyl ketones. I. Specificity of thrombin labeling.

In a new strategy for labeling the active sites of serine proteinases with fluorescence probes (Bock, P. E. (1988) Biochemistry 27, 6633-6639), a thioester peptide chloromethyl ketone inhibitor is incorporated into the enzyme active center and used to produce a unique thiol group which provides a site for selective chemical modification with any one of many thiol-reactive fluorescence probes. This approach was developed to increase the opportunities for identifying fluorescent proteinase derivatives that act as reporters of binding interactions by allowing a large number of derivatives, representing a broad range of probe spectral properties, to be readily prepared. In the studies described here, the specificity of the labeling approach was evaluated quantitatively for the labeling of human alpha and beta/gamma-thrombin with the thioester peptide chloromethyl ketones, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl and N alpha-[(acetylthio)acetyl]-D-Phe-Phe-Arg-CH2Cl, and the thiol-reactive fluorescence probe, 5-(iodoacetamido)fluorescein. Irreversible inactivation of thrombin by the inhibitors was accompanied by incorporation of 0.98 +/- 0.06 mol/mol of the thioester group into the active site, independent of a 470-fold difference between the thioester peptide chloromethyl ketones in the bimolecular rate constants of alpha-thrombin affinity labeling. Subsequent mild treatment of the covalent thrombin-inhibitor complexes with NH2OH in the presence of 5-(iodoacetamido)fluorescein resulted in generation of the thiol group together with its selective modification and incorporation of 0.96 +/- 0.07 mol of probe/mol of active sites. The incorporated label was localized to a 9000 molecular weight region of alpha and beta/gamma-thrombin containing the catalytic-site histidine residue. Evaluation of competing, side reactions showed that they did not significantly compromise the active site specificity of labeling. These results demonstrated equivalent, active-site-selective fluorescence probe labeling of alpha and beta/gamma-thrombin by use of either of the thioester peptide chloromethyl ketones, with a site specificity of greater than or equal to 94%.     

IDENTIFICATION OF THE CLASS PRYMNESIOPHYCEAE AND THE GENUS PHAEOCYSTIS WITH RIBOSOMAL RNA–TARGETED NUCLEIC ACID PROBES DETECTED BY FLOW CYTOMETRY1

Target regions specific for the class Prymnesiophyceae and the genus Phaeocystis (Har.) Lag. were identified from 18S ribosomal RNA coding regions, and two complementary probes were designed (PRYMN01 and PHAEO01). Detection of whole cells hybridized with these probes labeled with fluorescein isothiocyanate was difficult using epifluorescence microscopy because autofluorescence of the chlorophylls seriously interfered with the fluorescence of the probes. In contrast, flow cytometry proved very useful to detect and quantify the fluorescence of the hybridized cells. Hybridization conditions were optimized, especially with respect to formamide concentration. Both probes were tested on a large array of both target and nontarget strains. Positive and negative controls were also analyzed. Specificity was tested by adding a competing nonlabeled probe. Whereas probe PHAEO01 seems to have good specificity, probe PRYMN01 appeared less specific and must be used with stringent positive and negative controls.     

Development of a PNA probe for the detection of the toxic dinoflagellate Takayama pulchella

A peptide nucleic acid (PNA) probe was developed to detect the toxic dinoflagellate, Takayama pulchella TPXM, using fluorescent in situ hybridization (FISH) combined with epifluorescent microscopy and flow cytometry. The PNA probe was then used to analyze HAB samples from Xiamen Bay. The results indicated that the fluorescein phosphoramidite (FAM)-labeled probe (PNATP28S01) [Flu]-OO ATG CCA TCT CAA GA, entered the algal cells easily and bound to the target species specifically. High hybridization efficiency (nearly 100%) was observed. Detection by epifluorescence microscopy and flow cytometry gave comparable results. The fluorescence intensity of the PNA probe hybridized to T. pulchella cells was remarkably higher than that of two DNA probes used in this study and than the autofluorescence of the blank and negative control cells. In addition, the hybridization condition of the PNA probe was easier to control than DNA probes, and when applied to field-collected samples, the PNA probe showed higher binding efficiency to the target species than DNA probes. With the observed high specificity, binding efficiency, and detection signal intensity, the PNA probe will be useful for monitoring harmful algal blooms of T. pulchella.     

Fluorescent nanoparticles consisting of lipopeptides and fluorescein-modified polyanions for monitoring of protein kinase activity

Protein kinase (PK)-responsive nanoparticles (NPs) comprising a hydrophobically modified peptide substrate for PKs and a fluorescein-labeled polyanion (pA-F) were reported for monitoring PK activity via fluorescence intensity measurements. In this system, the formation of NPs by mixing lipopeptides and pA-Fs results in fluorescence quenching, while the quenched fluorescence recovered following dissociation of the NPs owing to the phosphorylation reaction of PKs. Eleven lipopeptides with different hydrophobic moieties (hydrocarbon and lithocholic acid) and four pA-Fs having main chains with differing flexibilities and fluorescein contents were synthesized and used to fabricate a series of twenty-four PK-responsive NP probes. The responses of the PK-responsive NP probes to PKs were evaluated to screen the most suitable NP probes. The assay system was then used to determine the IC(50) values for five inhibitors, the results of which were very similar to those previously reported. Thus, PK-responsive NPs are useful tools for high-throughput screening (HTS) of PK inhibitors.     

Fluorescence ratio measurements of double-labeled probes for multiple in situ hybridization by digital imaging microscopy.

To expand the multiplicity of the in situ hybridization (ISH) procedure, which is presently limited by the number of fluorochromes spectrally separable in the microscope, a digital fluorescence ratio method is proposed. For this purpose, chromosome-specific repetitive probes were double-labeled with two haptens and hybridized to interphase nuclei of human peripheral blood lymphocytes. The haptens were immunocytochemically detected with specific antibodies conjugated with the fluorochromes FITC or TRITC. The FITC and TRITC fluorescence intensities of spots obtained with different double-haptenized probes were measured, and the fluorescence ratio was calculated for each ISH spot. Combinations of different haptens, such as biotin, digoxigenin, fluorescein, sulfonate, acetyl amino fluorene (AAF), and mercury (Hg) were used. The fluorescence intensity ratio (FITC/TRITC) of the ISH spots was fairly constant for all combinations used, with coefficients of variation between 10 and 30%. To study the feasibility of a probe identification procedure on the basis of probe hapten ratios, one probe was double-labeled with different ratios, by varying the relative concentrations of the modified nucleotides (biotin-11-dUTP and digoxigenin-11-dUTP) in the nick-translation reaction. Measurement of the FITC and TRITC intensities of the ISH spots showed that the concentration of modified nucleotides used in the labeling procedures was reflected in the mean fluorescence intensity of the ISH spots. Furthermore, the ratio distributions showed little overlap due to the relatively small coefficients of variation. The results indicate that a multiple ISH procedure based on fluorescence ratio imaging of double-labeled probes is feasible.     

Biotin derivatives of methotrexate and folate. Synthesis and utilization for affinity purification of two membrane-associated folate transporters from L1210 cells

Biotin derivatives of methotrexate and folate (2-(biotinamido)ethyl-1,3'-dithiopropionyldiaminopentyl methotrexate and/or folate), in which carboxyl groups of the functional components are joined by a disulfide-containing spacer, have been synthesized, purified by DEAE-Trisacryl chromatography, and characterized by high pressure liquid chromatography and mass spectrometry. These bifunctional, dissociable probes were utilized for the single-step purification to homogeneity of two folate transport proteins (43 and 39 kDa) from L1210 cells. Treatment of the 39-kDa protein with peptide N-glycosidase F produced a smaller component (32 kDa); the 43-kDa protein, conversely, was unchanged by this procedure. When the 39-kDa transporter in intact cells was labeled with a fluorescein derivative of folate and then treated with phosphoinositol-specific phospholipase C, complete loss of fluorescence was observed. Alternatively, there was no change in fluorescence when the 43-kDa transporter was labeled with a fluorescein derivative of methotrexate and treated with the enzyme. These results indicate that the 43-kDa transporter is a nonglycosylated, integral membrane protein, whereas the 39-kDa counterpart is heavily glycosylated and anchored exofacially to the membrane by a glycosylphosphatidylinositol component.     

Active site selective labeling of serine proteases with spectroscopic probes using thioester peptide chloromethyl ketones: demonstration of thrombin labeling using N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl

The feasibility of a new approach to incorporation of spectroscopic probes into the active sites of certain serine proteases has been demonstrated. The method is based on inactivation of a serine protease with a thioester derivative of a peptide chloromethyl ketone. The thiol group generated by reaction of the covalent enzyme-inhibitor complex with NH2OH provides a unique site for subsequent labeling with thiol-reactive probes. To evaluate the method, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl was synthesized by reaction of the thrombin-specific tripeptide chloromethyl ketone with succinimidyl (acetylthio)acetate and purified by sulfopropyl-Sephadex and Sephadex G-10 chromatography. Reverse-phase high-performance liquid chromatography indicated that the product was 90 +/- 3% pure. The compound was quantitated by using 5,5'-dithiobis(2-nitrobenzoic acid) to measure the concentration of thiol produced in the presence of NH2OH. On this basis, titrations of the irreversible loss of human alpha-thrombin activity had end points of 1.1 +/- 0.1 mol of inhibitor/mol of active sites, indicating a 1:1 stoichiometry for inactivation. Incubation of N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-thrombin with 5-(iodoacetamido)fluorescein in the presence of NH2OH resulted in incorporation of 0.96 mol of the fluorescence probe/mol of active sites and the appearance of fluorescein fluorescence associated with the active site containing B-chain on sodium dodecyl sulfate-polyacrylamide gels. Fluorescence labeling of thrombin required reaction of the inhibitor at the active site as well as subsequent generation of the thiol group with NH2OH. It is concluded that active site selective labeling can be achieved by using this approach, which is likely to be applicable to other proteases, peptide chloromethyl ketones, and a wide variety of probes.     

Oligonucleotides labeled with single fluorophores as sensors for deoxynucleotide triphosphate binding by DNA polymerases

Oligonucleotides labeled with a single fluorophore (fluorescein or tetramethylrhodamine) have been used previously as fluorogenic substrates for a number of DNA modifying enzymes. Here, it is shown that such molecules can be used as fluorogenic probes to detect the template-dependent binding of deoxynucleotide triphosphates by DNA polymerases. Two polymerases were used in this work: the Klenow fragment of the Escherichia coli DNA polymerase I and the Bacillus stearothermophilus polymerase, Bst. When complexes of these polymerases with dye-labeled hairpin-type oligonucleotides were mixed with various deoxynucleotide triphosphates in the presence of Sr²⁺ as the divalent metal cation, the formation of ternary DNA-polymerase-dNTP complexes was detected by concentration-dependent changes in the fluorescence intensities of the dyes. Fluorescein- and tetramethylrhodamine-labeled probes of identical sequences responded differently to the two polymerases. With Bst polymerase, the fluorescence intensities of all probes increased with the next correct dNTP; with Klenow polymerase, tetramethylrhodamine-labeled probes increased their fluorescence, but the intensity of fluorescein-labeled probes decreased on formation of ternary complexes with the correct incoming nucleotides. The use of Sr²⁺ as the divalent metal ion allowed the formation of catalytically inactive ternary complexes and obviated the need for using 2′,3′-dideoxy-terminated oligonucleotides as would have been needed in the case of Mg²⁺ as the metal ion.     

Intracellular fusion of sequentially formed endocytic compartments

A polyclonal anti-fluorescein antibody (AFA) which quenches fluorescein fluorescence has been used to distinguish between two models of intracellular vesicle traffic. These models address the question of whether sequentially endocytosed probes will mix intracellularly or whether they are carried through the cell in a sequential, isolated manner. Using transferrin (Tf) as a recycling receptor marker, we incubated Chinese hamster ovary (CHO) cells with fluorescein-Tf (F-Tf) which is rapidly endocytosed. After the F-Tf was completely cleared from the surface, AFA was added to the incubation medium and entered endocytic compartments by fluid phase endocytosis. Fusion of a vesicle containing AFA with the compartment containing F-Tf results in binding of AFA to fluorescein and the quenching of fluorescein fluorescence. When AFA was added to the culture medium 2 min after clearance of F-Tf from the surface, time dependent fluorescence quenching occurred. After 20 min, 67% saturation of F-Tf with AFA was observed. When the interval between F-Tf clearance and AFA addition was increased to 5 min only 41% saturation of F-Tf was found. These data indicate that there are some compartments which are accessible for mixing with subsequently endocytosed molecules, but the efficiency of mixing falls off rapidly as the interval between pulses is increased. In CHO cells Tf swiftly segregates to a collection of vesicles or tubules in the para-Golgi region, and at steady state most of the F-Tf is in this compartment. Using digital image analysis to quantify quenching in this region, we have found that F-Tf/AFA mixing is occurring either within this compartment or before transferrin enters it.     

Fluorescent kabiramides: new probes to quantify actin in vitro and in vivo

We present the design, synthesis, and biochemical and spectroscopic characterization of five functional fluorescent conjugates of kabiramide C (KabC), a small molecule biomimetic of gelsolin. The tetramethylrhodamine (TMR), rhodol green (RG), IC5, dapoxyl (DAP), and fluorescein diester (FDE) conjugates of KabC bind specifically to actin at the barbed end in a 1:1 complex. These probes are shown to function in an indistinguishable manner to the unmodified KabC. Various modalities of the fluorescence emission of these KabC probes, including fluorescence anisotropy and fluorescence resonance energy transfer, are used for the development of assays for the rapid determination of G-actin concentration in solution. The TMR-KabC and FDE-KabC probes are cell permeable and provide unique imaging information on the distribution and dynamics of actin filament within living cells.     

The detergent Solulan C-24 reveals properties of the olfactory adenylate cyclase system.

We have been developing the use of plasma-membrane-bound fluorescent probes to measure the pH values at the surfaces of living chondrocytes. For this purpose, three lipophilic pH indicators were made by covalently binding the xanthene dyes fluorescein, eosin or dichlorofluorescein to the amino group of phosphatidylethanolamine. The probes were incorporated into phospholipid vesicles and the effect of pH on the fluorescence was characterized. Fluorescence was measured at a single emission wavelength during excitation at two wavelengths, and the ratio of the intensities was calculated. The experimentally observed pKobs. values were determined by fitting the fluorescence ratios to the Henderson-Hasselbalch equation. All three probes acted as pH indicators, and the eosinyl-, dichlorofluoresceinyl- and fluoresceinylphosphatidylethanolamines had pKobs. values of 3.5, 6.3 and 7.5 respectively. At physiological salt concentrations, changes in the composition of the vesicle membrane had little effect on these values. We concluded that these probes were promising candidates for the measurement of pH values at cell surfaces.