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PubMedDate not supplied

Neutral red supravital staining for cellular elements in the semen.

Human seminal fluid besides spermatozoa often contains other cellular elements. A supravital staining method designed to differentiate the above mentioned cellular elements was described. Amongst the spermatogenic cells only spermatocytes were stained with Neutral Red. They displayed two peculiar structures designated as "Y" granules and "Enigmatic Body". Neutral Red was absorbed by the spermiophage cells and was concentrated by them in the form of cytoplasmic granules. In addition the coarse granules of leukocytes and the gigestive vacuoles of Balantidium Coli and Trichomonads were stained with Neutral Red.

Eukaryota↗
PubMed1975

Failure of neutral-red photodynamic inactivation in recurrent herpes simplex virus infections.

Because photodynamic inactivation of herpes simplex virus infections may not be free of hazard, the efficacy of photodynamic inactivation with neutral red and light was evaluated in a placebo-controlled study of 170 episodes of recurrent herpes simplex virus infection in 96 patients. The preparation of neutral red that was used was shown to photoinactivate herpes simplex virus in vitro, but had no significant effect on the rate of resolution of herpetic lesions (P greater than 0.10) or on the frequency of subsequent recurrences (P greater than 0.10), except for orolabial lesions, in which an adverse effect on the rate of subsequent recurrences was observed (P less than 0.05). In the absence of demonstrated efficacy, the routine use of neutral red and light in patients with recurrent herpes simplex virus infections should be discontinued. Furthermore, other photoactive dyes should not be used until their efficacy has been demonstrated by suitably controlled clinical trials.

Administration, Topical↗
PubMed1976

Plaque reduction neutralization test for human cytomegalovirus based upon enhanced uptake of neutral red by virus-infected cells.

Foci of cells infected with human cytomegalovirus were noted to stain more intensely than uninfected cells with neutral red, and this provided the basis for development of a plaque assay and plaque reduction neutralization test for cytomegalovirus. Plaques demonstrable by neutral red staining could be counted at 8 days after infection; thus, results could be obtained earlier than for plaque assay systems based upon the viral cytopathic effect, a fewer manipulations were required for staining cell monolayers to demonstrate plaques. Certain variables affecting plaque size and numbers and antibody titers were defined. Addition of fresh guinea pig complement to the reaction mixtures markedly enhanced cytomegalovirus-neutralizing antibody titers of hyperimmune animal sera, but titers of human sera were enhanced only two-or fourfold.

Animals↗
PubMed1994

Application of neutral red staining for evaluation of the viability of dermatophytes and Candida in human skin scales.

The fungal elements observed by direct microscopic examination sometimes fail to grow on subsequent culture. To elucidate the cause of this discrepancy, the viability of dermatophytes and Candida in skin scales was evaluated by neutral red staining. Autoradiographic study using 3H-thymidine confirmed that grain-positive cells (viable cells) were stained with neutral red, whereas negative cells (non-viable cells) were not stained. Taking this as a baseline, the correlation between neutral red-positive fungal elements in scales taken from 211 patients with tinea and 27 patients with cutaneous candidosis and cultures on Sabouraud glucose agar was studied. Strong positive correlations were found in both mycoses. These findings suggest that neutral red staining provides a useful method for evaluating the viability of dermatophytes and Candida in human skin scales.

Arthrodermataceae↗
PubMed1977

[Influence of inhibitors of energy metabolism on the interaction of neutral red with Ehrlich ascitic carcinoma cells].

Effects of energy metabolism inhibitors on the granule formation of neutral red in Ehrlich ascite carcinoma cells have no relation with the action of these agents on the ATP formation. Accumulation of neutral red by cells has no relation with granule formation and does not depend on the energy metabolism of cells. The action of 2,4-dinditrophenol, oligomycin, Tween-20, Tween-80 and sodium deoxycholate on the granule formation may be linked with direct action of these agents on the cell membranes, for example, on the Golgi membranes.

Adenosine Triphosphate↗
PubMed1968

Enhancement of St. Louis arbovirus plaque formation by neutral red.

Experiments with St. Louis encephalitis (SLE) virus have shown that neutral red enhances the plaque size in duck embryo cell cultures. This may represent a new method for genetic studies of SLE virus population. In a mosquito pool specimen NR(+) and NR(-) particles were demonstrated. By intracerebral passage in mice, there is a selection of NR(+) particles. Similar effects were not shown for eastern, western, and California encephalitis viruses. Plaques formed by the latter virus, however, were significantly reduced in number by neutral red.

Animals↗
PubMed1981

[Neutral-red clearance technique for measuring gastric mucosal blood flow in the rat (author's transl)].

A neutral red (NR) clearance technique was described for measuring gastric mucosal blood flow in the rat. It is easily carried out, well reproducible and shows good correlation to results measured by other techniques. Our results suggest that the measurement of the neutral red clearance is directly related to the gastric mucosal blood flow. During the basal secretion, a mucosal blood flow of 0.1 ml-min-1 was measured in the anaesthetized stomach-perfused rat. Gastric mucosal blood flow (GMBF) increased up to 0.25 ml min-1 during a submaximal pentagastrin stimulation. In regard to the inhibition of acid secretion on ID50 of 2.04 micromol kg-1 i.v. was found for cimetidine. A dose of 2.54 micromol kg-1 i.v. of cimetidine halved the pentagastrin-induced increase of GMBF. Vasodilators increased the mucosal blood flow without influencing acid secretion.

Animals↗
PubMed1992

Distribution of intracellular pH in the rat brain cortex after global ischemia as measured by color film histophotometry of neutral red.

Tissue acidosis is an important determinant of cell viability following cerebral ischemia. Because of the heterogeneity of tissue response to metabolic stress, a method for measuring intracellular pH (pHi) that preserves spatial information would be desirable. Histophotometry of the pH indicator dye Neutral red offers such a possibility. The purpose of our study was to determine the distribution of pHi following complete irreversible ischemia and show the correlation of mean pHi measured by Neutral red and [31P]NMR in the same brain. Three rats were studied in the anesthetized state. A pHi range was obtained by total cerebral ischemia at various pre-arrest plasma glucose concentrations. The data show that mean pHi calculated by Neutral red was strongly correlated to pHi determined from [31P]NMR (slope: 0.99 +/- 0.08; P less than 0.001, r2 = 0.96). Within each brain, 80-110 discrete samples were analyzed by histophotometry. The pHi distribution of those samples broadened in those rat brains with greater acidosis, suggesting a heterogeneity of response by the tissue to ischemia and the presence of multiple pHi pools. Our results demonstrate the need to use methods which maintain spatial resolution such as is available with histophotometry.

Animals↗
PubMed1999

Neutral red assay of the cytotoxicity of fluorocarbon-coated polymethylmethacrylate intraocular lenses in vitro.

Polymethylmethacrylate (PMMA) intraocular lenses (IOLs) were coated with Teflon AF, an amorphous, transparent Teflon, to render them highly hydrophobic. Teflon-coated PMMA IOLs were immersed in culture medium for 30 days at 37 degrees C. Four concentrations of the IOL leachables, 2 concentrations of a toxic control (phenol), and complete liquid culture medium (nontoxic control) were incubated for 24 h in a 96-well plate containing confluent L-929 fibroblasts. The cytotoxic effect of each solution on the fibroblasts was quantitatively assessed by measuring the uptake of neutral red by the viable cells. After the extraction of the neutral red using 1% acetic acid-50% ethanol, the optical densities were measured with a microplate reader at 550 nm. Scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) were used to analyze the surfaces of the IOLs. Only the optical densities in the wells containing fibroblasts that had been in contact with the phenol solutions were significantly lower than those in the wells incubated with the nontoxic control solution (p < 0.01). There were no signs of surface alteration by SEM, apart from some crystals on the IOLs. The crystals were composed of Na and Cl, as demonstrated by XPS. Aqueous extractables from the Teflon-coated IOLs produced no cytotoxic effects in the neutral red assay used.

Animals↗
PubMed2005

Excretory/secretory products of sheep abomasal nematode parasites cause vacuolation and increased neutral red uptake by HeLa cells.

Excretory/secretory (ES) products of Ostertagia (Teladorsagia) circumcincta and Haemonchus contortus have been implicated in the inhibition of gastric acid secretion and vacuolation, and the loss of parietal cells associated with abomasal parasitism. Vacuolation of epithelial (HeLa) cells caused by adult O. circumcincta or L3 O. circumcincta or H. contortus ES products have been examined by differential interference contrast microscopy and by the neutral red uptake assay. ES products caused visible vacuolation of HeLa cells, and this effect was enhanced by 8 mM NH4Cl. Some parasite ES products caused a marked detachment of cells from the coverslip. At lower concentrations of ES products, neutral red uptake was usually increased above the control, but at higher concentrations of ES products, uptake was often decreased, probably because of cell detachment. Although generally consistent with direct observations of HeLa cell vacuolation by parasite chemicals, neutral red uptake was not a satisfactory quantitative assay.

Abomasum↗
PubMed1995

Discrimination of the toxic potential of chemically differing topical glucocorticoids using a neutral red release assay with human keratinocytes and fibroblasts.

In inflammatory skin disease, hydrocortisone and prednisolone double esters are about equipotent to conventional medium potency topical glucocorticoids, such as betamethasone valerate. Local adverse effects, in particular skin atrophy, are a potential problem with topical glucocorticoids. Recently, cell cultures have shown promise as a means of assessing local tolerance. To investigate the toxic potential of hydrocortisone, hydrocortisone-17-butyrate, hydrocortisone aceponate, prednicarbate, triamcinolone acetonide, betamethasone valerate and desoximethasone, human keratinocytes and fibroblasts were exposed to these agents in vitro, using a modified neutral red release assay. In addition, the morphology of these cells was assessed by light microscopy. Although all the topical glucocorticoids tested proved toxic to both cell types, there were major differences between glucocorticoids in their effect on fibroblasts. Hydrocortisone and the non-halogenated double-ester-type glucocorticoids were less toxic than the conventional medium potency topical glucocorticoids tested (betamethasone valerate and desoximethasone). In particular, hydrocortisone aceponate was less toxic than betamethasone valerate (P < or = 0.05). In general, the effect of topical glucocorticoids on the cells, based on neutral red release, was more marked with keratinocytes than with fibroblasts. Although the ranking order with respect to the toxic potential was similar, a clear-cut difference was not observed between non-halogenated double-ester-type glucocorticoids and betamethasone valerate. Morphological changes due to glucocorticoid exposure followed the same pattern with both keratinocytes and fibroblasts. The neutral red release assay is able to discriminate between the cytotoxic effects of chemically differing topical glucocorticoids on human keratinocytes and fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Topical↗
PubMed1990

Cytotoxic effects of food additives and pharmaceuticals on cells in culture as determined with the neutral red assay.

The acute cytotoxicity of butylated hydroxytoluene and butylated hydroxyanisole to cultured human dermal fibroblasts, keratinocytes, melanocytes, and melanoma tumor cells was determined with the neutral red assay. For all cell types, butylated hydroxytoluene proved to be more cytotoxic than butylated hydroxyanisole. The neutral red assay is a rapid, economical, semiautomated assay that can be used with a variety of cell types in culture to provide quantitated data that can be used to rank test agents according to their potencies.

Animals↗
PubMed1990

Usefulness of neutral red uptake method for investigation of the effects of recombinant hemopoietic growth factors on leukemic blasts proliferation.

By a colorimetric assay with dye (neutral red), the effects of recombinant human hemopoietic growth factors (rhG-CSF, rhGM-CSF, rhIL-3 and rhEPO) on the proliferation of leukemic blasts in vitro were investigated. Leukemic blasts were obtained from eight patients with acute myeloid leukemia (AML) (M1: two cases, M3: two cases, M4: four cases) and from four patients with acute lymphoid leukemia (ALL) (L2: four cases). It was shown that rhG-CSF, rhGM-CSF and rhIL-3 stimulated the blast proliferation in most cases of AML, although the degree and pattern of responses showed a striking variability in different patients. Moreover, rhGM-CSF and rhIL-3 also stimulated the leukemic blasts from some cases of ALL. No clear morphological modification providing evidence for terminal differentiation was observed when assessed by Wright stain. On the other hand, rhEPO did not have any stimulating effects on leukemic blasts in all cases. These results indicate the necessity of investigating the responses of leukemic blasts to hemopoietic growth factors in each patient prior to clinical use of such factors. For this assay, neutral red uptake method is useful because of its simplicity, rapidity, precision and convenience for handling a large volume of materials.

Adult↗
PubMed1976

Attachment of ribosomes to endoplasmic membranes in mouse pancreas. Degranulation in vivo caused by the inducers of autophagocytosis neutral red, vinblastine, puromycin, and cadmium ions, and prevention by cycloheximide.

The present investigation was undertaken to discover whether in vivo inducers of autophagocytosis such as neutral red (0.40 mg/g body weight), cadmium chloride (0.15 mg/animal), vinblastine sulfate (7.5 mg/kg b.w.), and puromycin dihydrochloride (0.20 mg/g b.w.) are able to produce degranulation of rough surfaced endoplasmic reticulum (ER) of pancreatic acinar cells as we suggested earlier. Using a modified method of Blobel and Potter about 30% of the total ribosomes of untreated control pancreas were recovered in the free form, and 70% in the membrane-bound form. Cycloheximide (0.20 mg/g body weight) had no effect on this distribution of ribosomes, while neutral red, cadmium ions, vinblastine, and puromycin led to the presence of more free ribosomes: thus up to 70% of the total cytoplasmic ribosomes were recovered in the free form 30 to 60 min after treatments with the autophagic inducers. Pretreatments with cycloheximide prevented this shift and the distribution of ribosomes remained normal. On the basis of these and previous results we conclude that in pancreas an initial degranulation of the ER caused by these agents is a precondition of membrane transformation in autophagocytosis which is preventable by cycloheximide. Confirming the ultrastructural data of Longnecker et al. on rat pancreas, puromycin was shown to cause different forms of cellular injury including autophagocytosis. All forms of cellular injury were also prevented by pretreatment with cycloheximide in mouse pancreas.

Animals↗
PubMed1975

The use of neutral red as a peroperative test of vagal innervation.

The cervical vagi were stimulated electrically in four groups of dogs after the intravenous injection of 10 ml of 1% neutral red. One group had intact vagi, the second group had denervation of the distal parietal cell area of the stomach, the third group proximal parietal cell denervation, and the last group had had a complete transthoracic truncal vagotomy. The area of stomach secreting neutral red was significantly related to the insulin-stimulated acid secretion. This technique may provide the basis of a test of residual vagal innervation of parietal cells during vagotomy operations in man.

Animals↗
PubMedDate not supplied

Comparison of cytotoxicity of various surfactants tested on normal human fibroblast cultures using the neutral red test, MTT assay and LDH release.

We used the neutral red test, MTT assay and lactate dehydrogenase (LDH) release to compare the potential cytotoxicity of six surfactants belonging to different classes--three non-ionic surfactants (Triton x100, octylphenoxypolyethoxy alcohol, from Orion; Tween 60, polyoxyethylene (20) sorbitan monostearate, from ICI Speciality Chemicals; Tween 80, polyoxyethylene (20) sorbitan monolaurate, from Labosi), two anionic surfactants (Texapon K1298, sodium lauryl sulphate, from Henkel; Texapon N40, sodium laurylether sulphate, from Henkel) and one cationic surfactant (benzethonium chloride, from Siber Hegner)--on human fibroblast cultures. According to the LC50 (microg ml(-1)), the tested surfactants can be classified in the following order of increasing cytotoxicity: Tween 80 < Texapon N40 < Tween 60 < Texapon K1298 < Triton x100 < benzethonium chloride.

Benzethonium↗
PubMed2005

Spectrometric and voltammetric investigation of interaction of neutral red with calf thymus DNA: pH effect.

The interaction of neutral red (NR) with calf thymus DNA (CT DNA) was investigated by spectrometric (UV-vis, circular dichroism and fluorescence) and voltammetric techniques. It was shown that the interaction of NR with DNA depended on the values of R (R is defined as the ratio of the concentration of NR to that of CT DNA) and pH of the solution. NR intercalated into CT DNA base pairs at lower R value (R < 2.4) and following by NR aggregating along the helical surface of DNA at higher R value (R > 2.4) in pH 6.0 solution. Interestingly, we found that at lower R value, NR intercalated into CT DNA with its long axis perpendicular or parallel to the dyad axis of DNA in the solution of pH 6.0. While in pH 7.0 solution, NR bound with CT DNA through intercalation and electrostatic interactions. The electrochemical inactive complexes, NR-2CT DNA, 3NR-CT DNA, and NR-CT DNA were formed when NR interacted with nucleic acids in pH 6.0 and 7.2 solutions, respectively. The corresponding intrinsic binding constants for these complexes were obtained by UV-vis and fluorescence spectrometric methods, respectively. The CD spectra showed that the conformation of CT DNA was converted from right-handed B-DNA to left-handed Z-DNA due to the aggregating of NR along the surface of DNA in pH 6.0 solution, whereas a conversion from B-DNA to C-DNA was induced due to the interaction of DNA with NR in pH 7.2 solution. Finally, two binding modes of NR with CT DNA in aqueous with different values of pH were shown in the scheme.

Animals↗
PubMed2000

Blastocystis hominis: neutral red supravital staining and its application to in vitro drug sensitivity testing.

A simple in vitro drug sensitivity testing system for Blastocystis hominis clinical isolates was developed. Application of supravital staining by neutral red allowed quantitative viability assessment. Four xenic cultures, isolated from human sources, were grown in modified monophasic Robinson's medium and tested for sensitivities to nine available drugs. Assessment was done using the cell-count method from air-dried preparations after supravital staining with neutral red. Also, the light absorbence method was evaluated. Trimethoprim, metronidazole, quinacrine, tetracycline, paromomycin, and two new antiprotozoal drugs, nitazoxanide and deacetyl-nitazoxanide, showed cytostatic or cytocidal effects on all or some Blastocystis isolates. Chloroquine and sulphamethoxazole did not demonstrate any effect at the concentrations studied.

Amebicides↗