Stomach scanning with radioiodinated toluilen red (neutral red).
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Animals↗
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Results for “Neutral Red”
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Utilization of electrically reduced neutral red by Actinobacillus succinogenes: physiological function of neutral red in membrane-driven fumarate reduction and energy conservation.
Neutral red (NR) functioned as an electronophore or electron channel enabling either cells or membranes purified from Actinobacillus succinogenes to drive electron transfer and proton translocation by coupling fumarate reduction to succinate production. Electrically reduced NR, unlike methyl or benzyl viologen, bound to cell membranes, was not toxic, and chemically reduced NAD. The cell membrane of A. succinogenes contained high levels of benzyl viologen-linked hydrogenase (12.2 U), fumarate reductase (13.1 U), and diaphorase (109.7 U) activities. Fumarate reductase (24.5 U) displayed the highest activity with NR as the electron carrier, whereas hydrogenase (1.1 U) and diaphorase (0.8 U) did not. Proton translocation by whole cells was dependent on either electrically reduced NR or H2 as the electron donor and on the fumarate concentration. During the growth of Actinobacillus on glucose plus electrically reduced NR in an electrochemical bioreactor system versus on glucose alone, electrically reduced NR enhanced glucose consumption, growth, and succinate production by about 20% while it decreased acetate production by about 50%. The rate of fumarate reduction to succinate by purified membranes was twofold higher with electrically reduced NR than with hydrogen as the electron donor. The addition of 2-(n-heptyl)-4-hydroxyquinoline N-oxide to whole cells or purified membranes inhibited succinate production from H2 plus fumarate but not from electrically reduced NR plus fumarate. Thus, NR appears to replace the function of menaquinone in the fumarate reductase complex, and it enables A. succinogenes to utilize electricity as a significant source of metabolic reducing power.
Actinobacillus↗
Evaluation of antifungal effects of terbinafine and itraconazole using neutral red staining.
Neutral red staining is a newly described method for evaluating the viability of fungal cells. Using this method, the antifungal effects of terbinafine and itraconazole were evaluated in vitro. When the number of neutral red-positive Trichophyton mentagrophytes conidia treated with these two oral antifungals was compared with the number of culture-proved viable T. mentagrophytes conidia, a strong positive correlation was found. This result confirms that neutral red staining is a useful method for evaluating the viability of T. mentagrophytes in vitro, and that it can be used for the evaluation of antifungal activity. We conclude that the effectiveness of an antifungal drug should not be evaluated by direct microscopic examination alone, but with the additional aid of mycological culture or neutral red staining.
Antifungal Agents↗
Photosensitization of herpes simplex virus type 1 with neutral red.
Commercial neutral red (NR) originally containing at least 8 components was purified by thin layer chromatography. Herpes simplex virus type 1 (HSV-1) treated in vitro with 30 microgram/ml of purified NR became sensitive to light inactivation within 2 min but rapidly lost this sensitivity upon dilution. Similarly, virus grown in the presence of NR lost its photosensitivity upon dilution of the virus stock. In both cases the kinetics of inactivation appeared to be multi-hit. Photoinactivation of intracellular virus was most effective when NR was applied between 6 and 12 h post-infection. The most efficient inactivation occurred when virus at pH 8.8 was irradiated by light at a wavelength of 470 nm.
Cell Line↗
The use of neutral red as an intracellular pH indicator in rat brain cortex in vivo.
Intracellular pH in the intact, normally perfused rat brain cortex was determined by rapid scanning reflectance spectrophotometry of Neutral Red. Neutral Red, a pH indicator dye, was administered intraperitoneally to rats. Reflectance spectra recorded from the exposed dural surface of 11 anesthetized rats were used to calculate an intracellular pH of 7.04 +/- 0.01. Detailed studies on the interactions of the dye with brain tissue were carried out in vitro to define the in vivo calibration curves. In addition, the physiological effect of dye administration on systemic blood pressure was determined, as well as uptake curves for Neutral Red into plasma and brain. It is concluded that Neutral Red can be used as an in vivo brain intracellular pH indicator and compares favorably with other methods of brain intracellular pH measurement with respect to accuracy, sensitivity, noninvasiveness, and stability and has the potential to exceed any existing method in time resolution.
Animals↗
The determination of cellular viability of hybridoma cells in microtitre plates: a colorimetric assay based on neutral red.
A colorimetric assay utilising Neutral Red (C.I. 50040), a nuclear stain, was developed to determine the cellular viability of hybridoma cells in microtitre plates. A linear correlation (r = 0.99) was found to exist between the uptake of Neutral Red by viable cells and the viable cell count determined by Trypan blue exclusion test. The linearity stretched over the range of cell concentrations normal in batch cultures (2-30 x 10(4)/0.2 ml) with as little as +/- 6% intra-plate well-to-well variation and +/- 10.2% inter-assay variation. Microscopical examinations of viable hybridoma cells stained with Neutral Red showed that it was located in the nucleus. The possible bifunctional activity of the Neutral Red assay as a test for cellular viability and estimating the DNA content of hybridoma cells is discussed along with its application in a drug screening programme.
Animals↗
The pharmacological relevance of vital staining with neutral red.
The uptake of neutral red into the renin-containing juxtaglomerular granules does not inhibit the release of renin either in basal or in stimulated states of renin secretion. The vasodilating effect of neutral red may be due to a nonspecific binding to noradrenaline-receptors in the vascular smooth muscle cells.
Angiotensin II↗
Quantitative in vitro assessment of phototoxicity by a fibroblast-neutral red assay.
We have adapted the neutral red uptake assay for quantitative assessment of injury to fibroblast cultures by potential phototoxins. Tetracycline derivatives, quinolone derivatives, and chlorpromazine were used as model compounds for development of the assay. Human fibroblasts were incubated with potential phototoxins, the cell cultures irradiated with UV, and the capacity for neutral red uptake determined. Demeclocycline and doxycycline, two known photosensitizers, showed a 94% and 95% decrease of neutral red uptake, respectively, indicating photo-induced cytotoxicity. Minocycline, a non-photosensitizing tetracycline derivative, showed no decrease in uptake. Tetracycline, a weak phototoxin, showed minor (10%) decrease at equivalent concentrations (20 micrograms/ml). Microscopic observation of neutral red uptake and cell damage paralleled the spectrophotometric findings. Chlorpromazine, a non-tetracycline phototoxin, showed 91% decrease. An additional group of phototoxic drugs, quinolone antibacterials, were studied. Nalidixic acid, ofloxacin, ciprofloxacin, and norfloxacin all demonstrated phototoxicity, with nalidixic acid showing the greatest decrease in neutral red uptake. This methodology may provide a useful rapid method to quantify phototoxic potential of new drugs or suspected phototoxins.
Cells, Cultured↗
Optical responses evoked by cerebellar surface stimulation in vivo using neutral red.
The pH sensitive dye, Neutral Red, was used with optical imaging techniques to map intracellular pH shifts elicited by cortical surface stimulation of the rat cerebellum. In the in vivo rat cerebellar cortex stained with Neutral Red, a brief stimulus train (three stimuli at 33 Hz) evoked a longitudinal beam of increased fluorescence (acidic shift) running parallel to the long axis of the folium within 100 ms of stimulation onset. A 5-10 s stimulus train (5-20 Hz) produced a biphasic optical response consisting of a beam of increased fluorescence (acidic shift) which returned to baseline in approximately 60 s, followed by a beam of decreased fluorescence (alkaline shift) for up to 120 s. A close spatial correspondence was observed between electrophysiological and optical maps of the response to surface stimulation. Application of acetazolamide enhanced the optical signals, acetabenzolamide-phenoxyethene had no effect, and the glutamate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, decreased the optical signals. Increased fluorescence was produced by superfusion of the cerebellar cortex with acidic Ringer solutions and a decrease in fluorescence by basic solutions. These fluorescence changes also occurred in the presence of several ion channel/receptor blockers. Increased fluorescence resulted from superfusion with Ringer solution containing sodium propionate and decreased fluorescence with the transition from 5% carbon dioxide to nominally carbon dioxide-free Ringer solution. Recovery from acid loading with ammonium chloride was prevented by amiloride, an inhibitor of the Na+/H+ transporter. Application of Ringer solution with high potassium concentration produced an increase in fluorescence but only a decrease in fluorescence was detected when neuronal blockers were present, an effect consistent with a glial contribution. This decrease in fluorescence was blocked by adding barium. No epifluorescent optical signals were obtained from unstained preparations or preparations stained with cell-permeant fluorescence markers, suggesting little contribution from activity-dependent volume changes and other intrinsic signals. These results demonstrate that the Neutral Red optical signals evoked by cerebellar surface stimulation are primarily pH based and include a significant component related to intracellular pH shifts. The large amplitudes of these optical signals are particularly useful for mapping neuronal activity. Furthermore, this technique provides a novel tool for the study of pH changes in vivo at both high spatial and temporal resolution.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
Neutral red as a hydrophobic probe for monitoring neuronal activity.
Neutral red fluorescence has been used to monitor neuronal activity. Local changes in either pH or hydrophobicity are reported to increase neutral red fluorescence, but the mechanisms underlying the increases remain unclear. In this study, the pH-dependent fluorescence changes in the basic and acidic forms of neutral red preloaded in rat cerebellar slices were separately measured with two excitation wavelengths. Bath application of kainate, domoate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) at 1 mM increased fluorescence of the acidic form and decreased that of the basic form irreversibly, consistent with a pH-dependent mechanism. In contrast, application of lower concentrations of AMPA, (S)-3,5-dihydroxyphenylglycine or KCl increased fluorescence of both forms transiently, suggesting that a pH-independent mechanism may also increase neutral red fluorescence. Electrical stimulation in the molecular layer also increased fluorescence of both forms. The response to weak electrical stimulation decayed in about 1 s, while response to intense stimulation lasted longer than 1 min. Neutral red binding to phospholipids was also detected as fluorescent spots on thin-layer chromatography, suggesting that interaction with phospholipids enhances neutral red fluorescence. Thus, the short-lasting signal of neutral red suggests its usage as a hydrophobic probe for neuronal activity.
Animals↗
Distinctive uptake of neutral red by mitotic cancer cells.
Neutral red stains both normal and cancer mitotic cells, but uptake by living mitotic cancer cells is distinctly higher than in normal cells. This new approach to cancer cell identification is demonstrated in 4 established tumorigenic cancer cell lines: human skin epidermoid carcinoma A431, mouse Cloudman malignant melanoma, human oral epidermoid carcinoma and rat hepatoma. Human Chang liver cells served as normal controls. With epidermal growth factor (EGF) prepulse, neutral red uptake is dramatically enhanced. The possibility of a causal relationship with M-phase specific phosphorylation is discussed.
Animals↗
DNA interaction, cytotoxicity, and radiosensitization with PtCl4(Nile Blue)2 and PtCl4(Neutral Red)2.
Complexes of the positively charged, nuclear staining, quinone-imine dyes Nile Blue and Neutral Red with negatively charged tetrachloroplatinum (II) have been prepared in an effort to form neutral drugs which could gain ready access to the cellular nucleus and deliver significant quantities of the reactive tetrachloroplatinum anion to the vicinity of the DNA. Elemental analysis showed that both the Nile Blue and Neutral Red complexes with tetrachloroplatinum (II) comprised 2 mol of dye and 1 mol of tetrachloroplatinum, forming Pt(Nile Blue)2 and Pt(Neutral Red)2. Exposure of superhelical pBR322 DNA to the complexes or the dyes for 24 h followed by agarose gel electrophoresis showed that Neutral Red and Pt(Neutral Red)2 had little effect on DNA conformation, but that both Nile Blue and Pt(Nile Blue)2 could produce single-strand DNA breaks in a dose-dependent fashion. Studies in exponentially growing asynchronous, hypoxic, and normally oxygenated EMT6 cells at normal pH (7.40) and pH 6.45 demonstrated that neither dye was highly toxic, but that both complexes were capable of producing significant cytotoxicity. Both complexes killed normally oxygenated cells more efficiently than hypoxic cells, but Pt(Neutral Red)2 was more cytotoxic at pH 6.45, while Pt(Nile Blue)2 killed significantly more cells at normal pH. Both complexes decreased the survival of hypoxic EMT6 cells as indicated by the slope of the radiation survival curve [dose modifying factor (DMF) 2.90 for Pt(Nile Blue)2 and 1.45 for Pt(Neutral Red)2]. Studies with the FSaIIC murine tumor showed that both complexes were active radiosensitizing agents in vivo [DMF 1.76 for Pt(Nile Blue)2 and 1.25 for Pt(Neutral Red)2]. These results indicate that these new platinum complexes have characteristics which may make them and similar complexes effective radiosensitizing agents in humans.
Animals↗
Electricity generation in microbial fuel cells using neutral red as an electronophore.
Neutral red (NR) was utilized as an electron mediator in microbial fuel cells consuming glucose to study both its efficiency during electricity generation and its role in altering anaerobic growth and metabolism of Escherichia coli and Actinobacillus succinogenes. A study of chemical fuel cells in which NADH, NR, and ferricyanide were the electron donor, the electronophore, and the electron acceptor, respectively, showed that electrical current produced from NADH was proportional to the concentration of NADH. Fourfold more current was produced from NADH in chemical fuel cells when NR was the electron mediator than when thionin was the electron mediator. In microbial fuel cells in which E. coli resting cells were used the amount of current produced from glucose when NR was the electron mediator (3.5 mA) was 10-fold more than the amount produced when thionin was the electron mediator (0.4 mA). The amount of electrical energy generated (expressed in joules per mole of substrate) and the amount of current produced from glucose (expressed in milliamperes) in NR-mediated microbial fuel cells containing either E. coli or A. succinogenes were about 10- and 2-fold greater, respectively, when resting cells were used than when growing cells were used. Cell growth was inhibited substantially when these microbial fuel cells were making current, and more oxidized end products were formed under these conditions. When sewage sludge (i.e., a mixed culture of anaerobic bacteria) was used in the fuel cell, stable (for 120 h) and equivalent levels of current were obtained with glucose, as observed in the pure-culture experiments. These results suggest that NR is better than other electron mediators used in microbial fuel cells and that sludge production can be decreased while electricity is produced in fuel cells. Our results are discussed in relation to factors that may improve the relatively low electrical efficiencies (1.2 kJ/mol) obtained with microbial fuel cells.
Actinobacillus↗
Ceramide stimulates the uptake of neutral red in human neutrophils, monocytes, and lymphocytes.
Neutral red is a vital stain known to be accumulated in the lysosomes of neutrophils and monocytes. It is used mainly to identify and detect the activated state of these cells. We have found that the extracellular application of physiological ceramide, i.e., a product of sphingomyelin hydrolysis and a newly defined intracellular second-messenger substance, increased the uptake of neutral red in a dose-dependent manner in human neutrophils, monocytes, and lymphocytes, as demonstrated by flow cytometry. Staurosporine was able to totally block this phenomenon, suggesting the involvement of protein kinase C in the process. These results indicate that the flow-cytometric analysis of ceramide-induced uptake of neutral red can be a new method for the evaluation of lysosome-related activation processes in both phagocytes and lymphocytes.
Arachidonic Acids↗
Solvent polarity and pH effects on the spectroscopic properties of neutral red: application to lysosomal microenvironment probing in living cells.
Neutral red is a lysosomal probe and a biological pH indicator. In aqueous solutions, the protonated (NRH) and neutral (NR) forms of monomeric neutral red exhibit distinct absorption maxima (535 and 450 nm, respectively) but have the same fluorescence with a maximum at 637 nm and a quantum yield of 0.02. The similarity of the fluorescence spectra at acidic and basic pH suggests deprotonation of cationic species in the first singlet excited state. The NR fluorescence strongly depends on the solvent polarity as shown by addition of increasing amounts of water to pure dioxane, which gradually shifts the fluorescence maximum from 540 nm in pure dioxane to 637 nm in water. The fluorescence quantum yield increases from 0.17 in dioxane to 0.3 upon addition of 7% water and then decreases, reaching 0.02 in pure water. Immediately after incubation of human skin fibroblasts with neutral red, excitation with 435 nm light produces a fluorescence whose maximum is recorded at 575 nm. This fluorescence is located in the perinuclear region and originates from large fluorescent intracytoplasmic spots, suggesting staining of the endoplasmic reticulum-Golgi complex. At longer times, this fluorescence is shifted to 606 nm, suggesting slow diffusion of the lysosomotropic dye toward the more hydrated and acidic interior of lysosomes. Addition of a lysosomotropic detergent to cells previously incubated with neutral red shifts the fluorescence to the blue. Thus, in complex biological systems, this probe cannot be a good pH indicator but is a very sensitive probe of lysosomal microenvironments.
Dioxanes↗
Neutral red staining for the assessment of acute outcome in rat focal cerebral ischemia models.
Neutral red staining was evaluated as an acute outcome assessment method in rat models of cerebral ischemia by comparison with histological infarction volume. Fischer 344 rats (n = 48) were used in three different models of middle cerebral artery (MCA) occlusion: proximal MCA occlusion (n = 16), distal MCA occlusion followed by ipsilateral common carotid artery (CCA) occlusion (distal MCA/CCA occlusion, n = 15), and MCA occlusion with an intravascularly introduced 4-0 nylon suture (intravascular MCA occlusion, n = 17). At 1 hour, 2 hours, and 4 hours after MCA occlusion, animals were injected with 2.5 ml of 4% neutral red solution via the femoral vein, and then sacrificed. Proximal MCA occlusion caused a neutral red defect volume in the cortex which correlated well with histological infarction volume at 4 hours (r = 0.88, p < 0.05), and in the caudate which correlated well with infarction volume at 4 hours (r = 0.94, p < 0.01). Distal MCA/CCA occlusion caused a neutral red defect volume in the cortex lager than the histological infarction volume (4 hrs: 88.6 +/- 11.8 vs. 74.3 +/- 17.4 mm3, p < 0.05) but closely correlated with the infarction volume at 4 hours (r = 0.81, p < 0.05). Intravascular MCA occlusion caused a neutral red defect volume in only two of 17 animals after 1-4 hours, which correlated well with the absence of histological evidence of infarction. Neutral red staining is a simple method for assessing the acute outcome of focal cerebral ischemia as early as 4 hours after the onset, in an appropriate model of cerebral ischemia.
Animals↗
The effect of in vitro heat stress on the uptake of neutral red by chicken thrombocytes.
Thrombocytes are multifunctional, nucleated blood cells. Morphological changes in thrombocytes have been used as a physiological indicator of a stress response. This study investigated the effects of in vitro heat stress (HS) on the neutral red uptake activity of chicken thrombocytes. Chicken thrombocytes (98% pure) were prepared from adult Barred Plymouth Rock and Rhode Island Red males. The isolated thrombocytes were preincubated at normal (41 C) or HS (45 C) temperatures for 30 min before either a 30- or 90-min incubation with neutral red at these same temperatures. After incubation the cells were washed, lysed, and the internal neutral red concentrations analyzed. There was no difference in thrombocyte numbers or their uptake of neutral red in samples from males of these two chicken breeds. At 41 C thrombocytes actively took up neutral red over the 90-min incubation period. However, at 45 C thrombocyte internalization of neutral red was significantly reduced. At both time periods (30 and 90 min), thrombocytes at 41 C took up significantly (P < or = 0.05) more neutral red than their counterparts at 45 C. This lack of neutral red uptake was not due to cell death as monitored by Trypan blue exclusion. Following the 30-min incubation there was no difference in viability between thrombocytes at the two temperatures. Although there was a significant (P < or = 0.05) increase in cell death at 90 min for thrombocytes kept at 45 C, the livability difference was of a much smaller magnitude than the difference in neutral red uptake when comparing to cells cultured at 41 C. Neutral red uptake is a rapid, inexpensive and repeatable technique for the study of thrombocyte function.
Animals↗
Ultrastructure of Trichophyton mentagrophytes stained with neutral red.
The ultrastructure of Trichophyton mentagrophytes cells stained with neutral red was investigated using electron microscopy and X-ray microanalysis. Fixatives containing molybdenum and chromium were used to prevent the outflow of neutral red. Electron-dense particles composed of metals and dye were observed exclusively in the vacuoles, which were increased in number and size, but not near the cell wall. Results indicate that neutral red passes directly through the fungal cell wall and is incorporated into the vacuoles.
Electron Probe Microanalysis↗