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Detection of viral surface antigens on HIV-2ben infected human tumor cell lines by flow cytometry.

The human monocytic cell line U-937 clone 2 and two T-cell lines CEM and MOLT-4 clone 8 were infected with HIV-2ben, a recent isolate of HIV-2. Infection and subsequent antigen expression on the cell surface was monitored by flow cytometry using a rabbit-anti-serum against tween-ether-treated HIV-2ben and a fluorescein-isothiocyanate-conjugated IgG against rabbit-IgG. The sensitivity of the three cell lines to infection with HIV-2ben correlated with the percentages of CD4-expressing cells but not with the levels of CD4-expression on the cell. The appearance of viral surface antigens preceded the formation of syncytia and correlated closely with the infecting virus dose. After 1-2 weeks in culture, 20-85% of the cells of each line expressed viral surface antigens. The variation depended on the cell type and cell culture conditions. The MOLT-4 clone 8 and the U-937 clone 2 cells died around 10 or 20 days, respectively, after HIV-2ben infection. Only HIV-2ben infected CEM cells grew permanently. Flow cytometry was an appropriate method to monitor the expression of viral proteins on the cell surface of HIV-infected cell lines. Flow cytometry proved to be more sensitive than determination of RT activity in supernatants of HIV-infected cells and more precise than light microscopy examinations.

Antigens, Surface↗

Chemosensitivity assay in mice prostate tumor: Preliminary report of flow cytometry, DNA fragmentation, ion ratiometric methods of anti-neoplastic drug monitoring.

Flow cytometry, DNA fragmentation, ion ratiomateric analysis and NMR peaks characterized drug chemosensitivity of antineoplastic drugs. Hypotheses were: 1. The chemosensitive effect of different cancer cell lines is characteristic; 2. DNA fragmentation, ion ratiometric analysis suggest apoptosis status of tumor cells. METHODS: PC-3 cell lines were compared with DU-145, LNCaP cell lines in culture for the [Na]i and [Ca]i ion sensing dyes, cell death, NMR peaks and apoptosis staining for chemotherapeutic action of different drugs. RESULTS: DNA fragmentation, ratiometric ions and fluorescence endlabelling plots were characteristic for cell lines and drug response. 31P-23Na NMR spectra showed characteristic high phospho-choline and sodium peaks. CONCLUSION: Flow cytometry, DNA fragmentation, ion ratiometric methods and NMR peaks indicated apoptosis and offered in vivo drug monitoring method.

Journal Article↗

Characteristics of different nucleic acid staining dyes for DNA fragment sizing by flow cytometry.

An efficient and reliable double-stranded DNA (dsDNA) staining protocol for DNA fragment sizing by flow cytometry is presented. The protocol employs 0.8 microM of PicoGreen to label a wide range of DNA concentrations (0.5 ng/mL to 10,000 ng/mL) without regard to the solution dye/bp ratios and without initial quantification of the DNA analyte concentration. Using a combination of spectrofluorometry and flow cytometry experiments, we found that PicoGreen exhibited better overall performance than all the tested dsDNA binding dyes, such as TOTO-1. Fluorometric titration revealed that typical DNA staining protocols designed on the basis of the dye/bp ratio were highly dependent upon the DNA concentration for optimal results. PicoGreen was the least sensitive to the solution dye/bp ratio and was highly fluorescent in the presence of dsDNA. Using this new protocol, accurate histograms of HindIII digested lambda DNA were demonstrated for DNA concentrations ranging from 5 to 2000 ng/mL, and for dye/bp ratios from 106:1 to 1:4 at 0.8 microM of PicoGreen. The new one-step protocol is broadly applicable to any sensitive, laser-induced fluorescence method for detection of nucleic acids.

Coloring Agents↗

DNA flow cytometry as a prognostic indicator in head and neck cancer.

The prognostic significance of deoxyribonucleic acid (DNA) flow cytometry has been investigated for many solid tumors, but few data have been accumulated for squamous cell carcinomas of the head and neck. To our knowledge, we report the largest number of patients (69) with head and neck primary carcinomas to be studied by DNA flow cytometry. In the first part of this study, we reviewed 109 consecutive patients with laryngeal or hypopharyngeal primary carcinomas which were treated at North Carolina Memorial Hospital during the period of 1981 to 1984. The final analysis comprised 139 DNA histograms (mean coefficient of variation: 8.02) on paraffin-embedded specimens from 48 patients. Of the 48 patients with primary carcinomas, 24 had glottic, 18 had supraglottic, and 6 had carcinomas from the piriform sinus. Patients had follow-up for a minimum of 12 months, with a mean follow-up period of 23 months. Twenty-three of the 48 primary carcinomas (48%) were clearly aneuploid, and the remaining 52% were tetraploid (22%) or diploid (30%). We have concluded that patients with clearly aneuploid primary carcinomas had significantly better prognoses than those with diploid tumors (p = 0.008). High DNA amounts (greater than 40% of cells beyond the diploid peak, DNA G1G0) also correlated with a favorable prognosis when compared with low DNA amounts (p less than 0.01), and this remained significant when the clinical outcome was adjusted for staging of the primary site (T), nodal status, and stage of disease. Ploidy was the most significant prognostic variable for the laryngeal group of patients. In the second part of the study, twenty-one patients with oral cavity squamous cell carcinomas were studied in a similar fashion as the group with laryngeal carcinomas. In this group, a low DNA amount, with 40% as the cutoff point, was associated with a favorable prognosis (p = 0.024), and this remained significant while controlling for T, nodal status, and stage of disease. Numbers were too small to permit evaluation of the impact of ploidy in this group, but there was a slight trend toward aneuploidy and tetraploidy, correlating with a poor treatment outcome (p = 0.228). DNA amount was the most significant prognostic variable for the group of patients with oral cavity carcinomas.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

Single-cell analysis of siRNA-mediated gene silencing using multiparameter flow cytometry.

BACKGROUND: Use of synthetic short interfering RNAs (siRNAs) to study gene function has been limited by an inability to selectively analyze subsets of cells in complex populations, low and variable transfection efficiencies, and semiquantitative assays for measuring protein down-regulation. Intracellular flow cytometry can overcome these limitations by analyzing populations at the single-cell level in a high-throughput and quantitative fashion. Individual cells displaying a knockdown phenotype can be selectively interrogated for functional responses using multiparameter analysis. METHODS: Lck-specific siRNA was delivered into Jurkat T cells or peripheral blood mononuclear cells (PBMCs) to suppress endogenous Lck expression. Transfected cells were fluorescently stained for intracellular Lck and analyzed using multiparameter flow cytometry. The Lck(lo) Jurkat subpopulation was selectively analyzed for CD69 up-regulation and phospho-states of signaling proteins following T-cell receptor (TCR) stimulation. Surface expression levels of CD4 and CD8 on transfected CD3+ gated PBMCs were correlated with intracellular Lck levels. RESULTS: A subpopulation of Jurkat cells with reduced levels of Lck was clearly resolved from cells with wildtype levels of Lck. Both CD69 up-regulation and ZAP70 phosphorylation were suppressed in Lck(lo) cells when compared with those in Lck(hi) cells upon TCR stimulation. Knockdown of intracellular Lck in primary T lymphocytes reduced surface expression of CD4 in a dose-dependent manner. CONCLUSIONS: Multiparameter flow cytometry is a powerful technique for the quantitative analysis of siRNA-mediated protein knockdown in complex hard-to-transfect cell populations.

Antigens, CD↗

[Diagnostic importance of flow cytometry in staging malignant lymphoma].

Bone marrow biopsy plays an important role in the clinical diagnosis of malignant lymphoma. Further diagnostic methods need to be established to increase accuracy in the light of advances over recent years in the immunophenotypical characterization of discrete lymphatic bone marrow lesions and the continuing difficulty in classifying them. This study compared the diagnostic value of flow cytometry to that of conventional bone marrow biopsy in 156 patients with 191 marrow biopsy specimens or bone marrow aspirates. The most important findings were that up to one-third of lymphomas could not be diagnosed by flow cytometry, and that the degree of infiltration was estimated as less than two-thirds. However, flow cytometric results were more satisfactory in acute lymphoblastic leukemia, lymphoblastic lymphoma, hairy cell leukemia, and chronic lymphocytic leukemia. In summary, flow cytometry has a complementary role in the staging of lymphoma but cannot fully replace conventional trephine biopsy.

Biopsy↗

Ploidy, proliferative activity and prognosis. DNA flow cytometry of solid tumors.

Flow cytometric study has been used to measure the cellular DNA content of solid tumors for the last decade, and of paraffin-embedded tumor specimens for the last 5 years. Ploidy and proliferative activity are the two properties commonly measured by DNA content flow cytometric study. The ability to study archival, paraffin-embedded tumors has hastened an appreciation of the prognostic utility of this assay. Either abnormal ploidy or elevated proliferative activity predict a worsened disease-free or overall survival in most common adult malignancies. Both abnormalities are associated with poor outcome in locoregional breast, non-small cell lung, and colorectal cancers, and in all stages of ovarian cancer. Abnormal ploidy is also a dire prognostic indicator for cancers arising from the kidney, bladder, prostate, and endometrium. Clinical management of patients with these diseases may be aided by studying their tumors for these objective markers of biological aggressiveness.

DNA, Neoplasm↗

Evaluation of methods for measuring cellular glutathione content using flow cytometry.

The currently available flow cytometric stains for cellular glutathione were evaluated, examining the labelling of both human and rodent cell lines under various conditions of concentration, time, and temperature. Procedures were used that depleted glutathione (GSH) while having a minimal effect on other cellular sulphydryls in order to estimate linearity and the extent of background staining. As previously reported, monochlorobimane was highly specific for GSH in rodent cells but failed to label human cells adequately because of its low affinity for human glutathione S-transferases. Higher concentrations of monochlorobimane achieved more complete labelling of the human cellular GSH pool but gave increased background fluorescence due to non-GSH binding. The analogue monobromobimane, which binds nonenzymatically to sulphydryls, reacted more readily with GSH than with protein sulphydryls and, provided that stain concentration and incubation time were controlled, gave reproducible staining of human cells with approximately 20% of total fluorescence due to background staining. Of the currently available stains for measuring GSH in human cells, monobromobimane is the agent of choice. Mercury orange also binds more readily to GSH than to protein, giving a degree of specificity, and it has the additional advantage of being excited at 488 nm. However, the reproducibility of staining with mercury orange was less consistent than that using monobromobimane, and a higher background fluorescence was seen. Two additional stains, o-phthaldialdehyde and chloromethyl fluorescein, could also be used to label cellular GSH, but both gave an unacceptably high level of background staining. It is recommended that flow cytometric GSH assays should routinely include a sample of cells that have been depleted of GSH in order to determine the extent of background labeling.

Animals↗

Mitotic cell cycle control in Physarum. Unprecedented insights via flow-cytometry.

High resolution flow-cytometric studies of isolated macroplasmodial nuclei of the myxomycete. Physarum polycephalum provide definite evidence for the persistence of natural synchrony at mitosis throughout the entire cell cycle, i.e. completely synchronous DNA replication and traverse of G2. Even if nuclei within a given macroplasmodium belong to two distinct genome size classes (mixoploidy), they cycle and traverse mitosis in strict synchrony. This cannot be explained by current models of regulation of division based solely upon nuclear size and/or nuclear/cytoplasm ratios. Constitutional DNA content variation was apparent among all tested strains, and loss of late-replicating, presumably AT-rich DNA accounts for this variation. A constant duration of the S phase is maintained, irrespective of DNA content, via differential slowdown of replication rates during the 2nd and 3rd hours of replication. A frequently described extension of nuclear replication into G2 could not be substantiated. Interference with DNA and protein synthesis provides the first evidence for a brief "G1 phase" equivalent of 3-4 min duration in asynchronous microplasmodial cultures, and temporally assigns a protein synthesis-dependent "transition point" for completion of mitosis and initiation of DNA synthesis at 5 min prior to actual division nuclei which have passed this point at the time of addition of cycloheximide replicate 5% of their DNA before they become arrested. These findings provide strong experimental support for the transition point concept of cell cycle control, and additionally are commensurate with some form of the replicon-set hypothesis in Physarum.

Cell Nucleus↗

Metal-induced alteration of the cell membrane/cytoplasm complex studied by flow cytometry and detergent lysis.

Flow cytometric analysis of the cell cycle is most effectively accomplished with membrane-/cytoplasm-free ("clean") nuclei. Non-ionic detergents (e.g. NP40 or Triton X-100) commonly are employed to solubilize cell membranes/cytoplasm to produce "clean" nuclei. Treatment of murine erythroleukemic cells (MELC) with tri-n-butyltin methoxide, cadmium acetate, zinc sulfate, or lead acetate alters the properties of the cell membrane/cytoplasm complex making it resistant to NP40 dissolution. On a molar basis, the organotin compound was more effective in inducing resistance to detergent-mediated dissolution than the inorganic metal compounds. Resistance to NP40-mediated dissolution was manifested as an increase in the flow cytometric parameters 90 degrees scatter and fluorescein isothiocyanate (FITC) fluorescence and was confirmed by light microscopy.

Acetates↗

Discrimination of damaged/dead cells by propidium iodide uptake in immunofluorescently labeled populations analyzed by phase-sensitive flow cytometry.

We report a flow cytometric fluorescence lifetime-based method to discriminate damaged/dead from viable cells in immunofluorescently labeled populations using propidium iodide as a dye-exclusion viability probe. Fluorescence signals from propidium iodide and the anti-thymus cell-surface immunofluorescence marker fluorochromes, phycoerythrin and phycoerythrin/Texas Red (tandem conjugate), which have overlapping emission spectra with propidium iodide, are resolved based on differences in their fluorescence emission lifetimes using phase-sensitive detection. Mouse thymus cell samples were first labeled separately with anti-Thy 1.2 antibody directly conjugated to phycoerythrin and to phycoerythrin/Texas Red and propidium iodide. Labeled cells were then analyzed to determine the lifetimes of the immunofluorescence markers and propidium iodide. Based on these results, rat and mouse thymocytes labeled with anti-Thy 1.1 conjugated to phycoerythrin and anti-Thy 1.2 conjugated to phycoerythrin/Texas Red, respectively, were suspended in phosphate buffered saline containing propidium iodide, and were analyzed as they passed through a flow chamber and crossed a high-frequency, intensity-modulated (sinusoidal) laser excitation beam. The resulting immunofluorescence and propidium iodide signals were resolved based on differences in fluorescence lifetimes expressed as phase shifts using phase-sensitive detection and displayed as frequency distribution histograms and bivariate contour diagrams. This technology provides a new method to resolve immunofluorescence and propidium iodide signals from overlapping fluorescence emission spectra and a flow cytometric lifetime-based technique to quantify damaged/dead cells in immunofluorescence studies.

Animals↗

Analysis of nuclear DNA content in Capsicum (Solanaceae) by flow cytometry and Feulgen densitometry.

Flow cytometric measurements of nuclear DNA content were performed using ethidium bromide as the DNA stain (internal standard, Hordeum vulgare 'Ditta', 1C = 5.063 pg) in 25 samples belonging to nine diploid species and four varieties of Capsicum: C. chacoense, C. parvifolium, C. frutescens, C. chinense, C. annuum var. annuum, C. baccatum var. baccatum, C. baccatum var. pendulum, C. baccatum var. umbilicatum, C. eximium and C. pubescens, all with 2n = 24, and C. campylopodium with 2n = 26. In addition, one sample each of C. annuum var. annuum and C. pubescens were also analysed using Feulgen densitometry (standard, Allium cepa 'Stuttgarter Riesen', 1C = 16.75 pg). Both staining methods resulted in very similar relative values. Genome size displays significant variation between but not within species (except in C. campylopodium), and contributes to their taxonomic grouping. 1C-values range from 3.34-3.43 pg (3273-3361 Mbp) in C. chacoense and the C. annuum complex to 4.53-5.77 pg (4439-5655 Mbp) in C. campylopodium and C. parvifolium. The data obtained support conclusions on phylogenetic relationships in the genus derived from karyotype analyses using chromosome banding approaches. In Capsicum, constitutive heterochromatin amount is correlated with genome size, except in C. parvifolium, and is regarded as an additive genomic component.

Capsicum↗

Comparative scoring of micronucleated reticulocytes in rat peripheral blood by flow cytometry and microscopy.

A flow cytometric technique for scoring the incidence of micronucleated reticulocytes in rat peripheral blood was compared to a standard microscopy-based procedure. For these studies, groups of five male Sprague-Dawley rats were treated with vehicle or a broad range of chemical genotoxicants: 6-thioguanine, N-methyl-N'-nitro-N-nitrosoguanidine, vincristine, methylaziridine, acetaldehyde, methyl methanesulfonate, benzene, monocrotaline, and azathioprine. Animals were treated once a day for up to 2 days, and peripheral blood was collected between 24 and 48 h after the final administration. These samples were processed for flow cytometric scoring and microscopy-based analysis using supravital acridine orange staining, and the percentage of reticulocytes and micronucleated reticulocytes was determined for each sample. The resulting data demonstrate good agreement between these scoring methodologies, although careful execution of the flow cytometric method was found to enhance the micronucleus assay by reducing both scoring time and scoring error. These data add further support to the premise that the peripheral blood compartment of rats can be used effectively to detect genotoxicant-induced micronuclei.

Acridine Orange↗

Tracking and characterisation of transfused platelets by two colour, whole blood flow cytometry.

We describe a flow cytometric technique to study transfused platelets in patients. By selecting donor/recipient pairs discrepant for HLA-A2, transfused platelets were identified using anti-HLA-A2 with a detection limit of <5%, and accuracy within 4.3% of predicted (r2 > 0.96, P < 0.0001). In two of three episodes, transfused platelets were present in greater numbers than predicted from increment counts. Platelets with bound fibrinogen fell from 20.9 +/- 23.6% of donor platelets pretransfusion to 1.7 +/- 0.3% 1 h post-transfusion, whereas P-selectin-positive platelets fell gradually, from 24.1 +/- 6.7 to 7.3 +/- 3.3% at 6 h. This method avoids radio or chemical labelling, and could be used to assess novel platelet preparations post-transfusion.

Antibodies, Monoclonal↗

Quantitative determination of the number of FITC-molecules bound per cell in immunofluorescence flow cytometry.

Results obtained by flow cytofluorometry are expressed in 'arbitrary' units, which have a relative significance only. A method is described to make this unit to represent a number of fluorochrome molecules per cell, i.e. to give it an absolute value. The method makes use of an antibody labeled with a fluorochrome (FITC) as well as a radioactive marker (125I). From the amount of radioactivity bound per cell on the one hand and the fluorescence measured by flow cytofluorometry on the other, the amount of FITC per unit of fluorescence follows by straight-forward calculation. The method described is rather simple to perform and is quite reliable. Some of its restrictions are discussed.

Cell Separation↗

Functional opsonic activity of human serum antibodies to inner core lipopolysaccharide (galE) of serogroup B meningococci measured by flow cytometry.

A recently described flow cytometric opsonophagocytic assay (OPA) was adapted to quantify the functional activity of serum antibodies specifically directed against serogroup B inner core lipopolysaccharide (LPS) of Neisseria meningitidis. The percentage of human peripheral polymorphonuclear leukocytes and monocytes (PMNms) ingesting fluorescently labeled, ethanol-fixed N. meningitidis organisms (phagocytic activity) in the presence of human sera was measured to reflect the serum opsonic activity against the bacterium. The contribution to opsonophagocytic activity of antibodies to inner core LPS was estimated by comparing the opsonic activities of adult and infant sera before and after adsorbing anti-LPS antibodies from the sera using purified LPS extracted from an LPS mutant (galE) of N. meningitidis strain MC58 (B:15:P1.7,16:L3). The specificity of the assay was further investigated using monoclonal antibody (MAb) B5, which binds to an inner core LPS epitope of N. meningitidis. A dose-dependent decrease in phagocytic activity was observed when MAb B5 was incubated with LPS from an inner core LPS (galE) mutant. Similarly, the number of PMNms ingesting fluorescently labeled polystyrene beads coated with inner core (galE) LPS decreased in a dose-dependent fashion when MAb B5 was incubated with various concentrations of the homologous inner core LPS. Strong correlations were found between the concentration of serum antibodies to inner core LPS (galE) versus the phagocytic activity using healthy adult sera (r(2) = 0.89). There was a correlation between phagocytic ingestion and initiation of intracellular oxidative burst (r(2) = 0.99) using polystyrene beads coated with inner core LPS and opsonized with the same sera using the oxidative burst indicator system dihydrorhodamine123/rhodamine 123. OPA results were also found to correlate closely with the results of the serum bactericidal assay using MAb B5 against the N. meningitidis MC58 galE mutant in the presence of human complement (r(2) = 0.994, P = 0.003, two-tailed test). These studies demonstrate that functional antibodies are produced in humans against meningococcal inner core LPS and that the OPA is a useful approach to study the opsonic activity of antibodies to inner core LPS in health and disease.

Adult↗

Diagnosis of molar pregnancy and persistent trophoblastic disease by flow cytometry.

Histopathological assessment and flow cytometric analyses were carried out on 32 placentas (representative of each trimester) and 88 molar pregnancies. Three first trimester placentas were triploid, and the remaining 29 placentas were diploid. Of the 88 cases originally diagnosed as molar pregnancies, 26 were triploid (two complete moles, 20 partial moles, and four hydropic abortions); 59 were diploid (46 complete moles, 10 partial moles, three hydropic abortions); one was tetraploid (partial mole); and two were DNA aneuploid (one partial mole, one complete mole). A significantly increased hyperdiploid fraction (a measure of cell proliferation) was detected in diploid complete moles (p less than 0.0001) and cases of persistent trophoblastic disease (p less than 0.001) when compared with diploid placentas and diploid partial moles. All seven cases of established persistent trophoblastic disease, for which follow up was available, were diploid and showed high hyperdiploid fractions within the range for diploid complete moles. These findings suggest that flow cytometric DNA measurements may be an important aid in the diagnosis of molar pregnancy. The high degree of cell proliferation found in this study may explain the premalignant potential of complete hydatidiform moles.

Diploidy↗

Bone marrow cell differential counts obtained by multidimensional flow cytometry.

Five-dimensional flow cytometric analysis of normal bone marrow aspirates was utilized to determine the frequency of neutrophils, eosinophils, monocytes, lymphocytes, nucleated erythrocytes, reticulocytes, platelets, and a cell population that included blasts of each of the cell lineages, megakaryocytes, plasma cells, and basophils. Each of these bone marrow cell populations had unique features with respect to forward light scatter, orthogonal light scatter, and staining with Thiazole-Orange, LDS-751, and CD45 labeled with Phycoerythrin (PE). The identity of the cell populations was verified by sorting each of the cell populations and subsequent light microscopic examination of the cells. The frequencies of the nucleated bone marrow cell subpopulations of 50 normal donors were for neutrophils, mean 72.3%; SD +/- 5.1; 95% limits, 70.9-73.8%; eosinophils, mean 1.8%; SD +/- 1.3; 95% limits, 1.4-2.1%; monocytes, mean, 2.8%; SD +/- 1.2; 95% limits, 2.5-3.1%; lymphocytes, mean 12.1%; SD +/- 3.6; 95% limits 11.1-13.2%; nucleated erythrocytes, mean 8.9%; SD +/- 3.9; 95% limits, 7.8-10.1%; and the cell population that included blasts of each of the cell lineages, megakaryocytes, plasma cells, and basophils, mean 1.6%; SD +/- 1.2; 95% limits, 1.3-1.9%. The percentage of reticulocytes in bone marrow aspirates from 50 normal donors correlated with the reticulocyte frequency in the peripheral blood of these donors. However, the mean frequency of reticulocytes was significantly greater (p < 0.0001) in bone marrow (mean 2.19%; SD +/- 0.88) than in peripheral blood (mean 1.71%; SD +/- 0.88). The technique could discriminate between immature and mature reticulocytes based on the brighter staining with both Thiazole-Orange and LDS-751 of the immature reticulocytes. This was confirmed by cell sorting of both reticulocyte populations, which revealed larger clumps of New Methylene Blue staining material in the brighter Thiazole-Orange and LDS-751 stained reticulocytes. The immature reticulocytes were present in normal bone marrow, but not in normal peripheral blood. As expected, a significantly greater frequency of nucleated cells was found in bone marrow aspirates (mean 0.85%; SD +/- 0.59) than in peripheral blood (mean 0.20%; SD +/- 0.11). The frequency of platelets was significantly lower in bone marrow (mean 1.24%; SD +/- 0.69) than in peripheral blood (mean 2.94%, SD +/- 1.14). Flow cytometric bone marrow analysis can provide clinical laboratories with a technique that generates quantitative bone marrow cell differentials and potentially can reduce the need for light microscopic examination of bone marrow smears.

Benzothiazoles↗