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Results for “Neutral Red”

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PubMed1995

Interferon-gamma inhibits endocytosis of soluble animated beta-1,3-D-glucan and neutral red in mouse peritoneal macrophages.

We previously demonstrated that soluble animated beta-1,3-D-glucan (AG) is internalized after binding to a specific beta-glucan receptor on macrophages. Internalization, but not binding, of AG is reduced when the macrophages are treated with IFN-gamma. Because our data indicated that AG is taken up by macrophages through beta-glucan receptor-mediated endocytosis, we wanted to characterize further the inhibitory effect of IFN-gamma on endocytosis. We compared the internalization of AG and neutral red (NR). NR is internalized by macrophages through fluid-phase endocytosis. AG and NR showed a similar influx/efflux pattern. The initial rate of accumulation was much larger for AG than for NR, however, probably because of the involvement of the beta-glucan receptor in the uptake of AG. Internalized AG was associated with membranes of the endocytic vesicles and formed characteristic rings on confocal laser scanning microscopy (CLSM) images. Both the influx and efflux of AG and NR was inhibited by treatment of macrophages with IFN-gamma. Phorbol myristate acetate (PMA) added to the cell cultures increased the accumulation of AG and NR and reversed the inhibitory effect of IFN-gamma. The effect of PMA was dependent on functionally intact microfilaments and microtubules. CLSM showed that the accumulated AG was localized mostly in small vesicles (size < 2 microns) in IFN-gamma-treated cells, in large and small vesicles in untreated cells, and mostly in large vesicles (size > 2 microns) in PMA-treated cells. In conclusion, IFN-gamma inhibits both the beta-glucan receptor-mediated endocytosis of AG and the fluid-phase endocytosis of NR, probably by inhibiting the formation of large vesicles.

Amination↗
PubMed2005

Assessment of the phototoxic hazard of some essential oils using modified 3T3 neutral red uptake assay.

When substances are developed in the aim to be a constituent of personal care products, and to be applied on the skin, it is necessary to carry out an assessment of potential phototoxic hazard. Phototoxicity is skin reaction caused by concurrent topical or systemic exposure to specific molecule and ultraviolet radiation. Most phototoxic compounds absorb energy particularly from UVA light leading to the generation of activated derivatives which can induce cellular damage. This type of adverse cutaneous response can be reproduced in vitro using different models of phototoxicity such as the validated 3T3 Neutral Red Uptake (NRU) phototoxicity assay. In the present study we utilised two different cell lines (the murine fibroblastic cell line 3T3 and the rabbit cornea derived cell line SIRC) to compare the photo-irritation potential of a strong phototoxic compound, chlorpromazine, to a weaker composite, such as 8-methoxypsoralen and Bergamot oil. After comparison of the different systems, five other essential oils were tested with both cell lines. Cellular damage was evaluated by the NRU cytotoxicity test or by MTT conversion test.

Animals↗
PubMed2004

A review of lysosomal membrane stability measured by neutral red retention: is it a workable earthworm biomarker?

The ability of biomarkers to integrate effects of chemicals on biota has lead to increased calls for their application in assessing the status of polluted ecosystems. In tandem there has been an increase in our knowledge of the ecophysiological responses of keystone species to pollutants, which has allowed the development of a number of promising methods. In contrast to the number of biomarker development studies, the number of biomarker validation studies has remained limited. This paper redresses this imbalance by drawing together data from studies that have used the earthworm lysosomal membrane stability response (measured using the neutral red retention assay). This review first gives a short history of the biomarker's development. Second, it sets published applications of the technique against established criteria for a "good" biomarker (i.e., dose-response relationship, sensitivity, ecological relevance, confounding factors, chemical specificity, species differences, time-response relationship, methodological concerns, and overall public/regulator confidence, and acceptance). Discussion of the biomarker's suitability to each criterion is followed by an overall evaluation of its workability for routine soil quality assessment and caveats for its use.

Animals↗
PubMed2001

Cytotoxicity of dental glass ionomers evaluated using dimethylthiazol diphenyltetrazolium and neutral red tests.

The purpose of this study was to assess the cytotoxicity of some commonly used glass ionomers. Three chemically cured glass ionomers (Fuji II, Lining cement, and Ketac Silver) and one light-cured (Fuji II LC) were tested. Extracts of mixed non-polymerized materials and polymerized specimens were prepared in accordance with ISO standard 10993-12. The polymerized specimens were cured and placed either directly in the medium (freshly cured), left for 24 h (aged), or aged plus ground before being placed in the medium. The cytotoxicity of extracts was evaluated on mouse fibroblasts (L, 929), using dimethylthiazol diphenyltetrazolium (MTT) and neutral red (NR) assays. Further, the concentrations of aluminum, arsenic and lead were analyzed in aqueous extracts from freshly cured and aged samples, and the fluoride levels analyzed in aqueous extracts from freshly cured samples. All extracts except that of non-polymerized Ketac Silver were rated as severely cytotoxic in both assays. Extracts of polymerized material were significantly more cytotoxic than extracts of non-polymerized material. All freshly cured glass ionomers released aluminum and fluoride concentrations far above what is considered cytotoxic (aluminum >0.2 ppm and fluoride >20 ppm). Extracts from freshly cured Lining Cement contained the highest concentrations of aluminum and fluoride (215 ppm and 112 ppm). Extracts from freshly cured Ketac Silver had the lowest concentrations of aluminum and fluoride but the highest of lead (100 ppm). It can be concluded that all extracts from non-cured, freshly cured, and aged glass ionomers contained cytotoxic levels of substances. Curing did not reduce the toxicity significantly.

Aluminum↗
PubMed1989

Neutral red uptake and clonogenic survival assays of the hyperthermic sensitization of tumor cells to tumor necrosis factor.

Vital dye uptake and postfixation dye assays have recently been used to examine the interaction between short-term (24-48 h) exposures to the monokine, tumor necrosis factor (TNF), and hyperthermic treatments with the finding that synergistic increases in cytotoxicity occurred. However, survival measured by these short-term dye assays is not necessarily closely related to eventual loss of clonogenic capacity. Treatment-induced growth delays, delayed cytotoxic effects, or perturbations of vital dye sequestration mechanisms could result in a different measurement of surviving fraction than given by a clonogenic assay. In this study we directly compared the neutral red vital dye uptake and clonogenic survival assays and confirmed in both assays that TNF-sensitive (L-929) and TNF-resistant (EMT-6) phenotypes show greatly reduced survival when treated with combined recombinant human TNF (1.0-0.0005 micrograms/ml) and hyperthermia (1-2 h at 43 degrees C). Moreover, we confirmed that sensitization of the TNF-resistant EMT-6 cells was largely dependent on monokine treatment before hyperthermia and was reduced by the reverse sequence. The greatest sensitization of TNF-responsive L-929 cells also occurred when TNF treatment preceded heating. These results for clonogenic survival are consistent with the hypothesis that hyperthermia used in combination with TNF in vivo is more cytotoxic than TNF or hyperthermia separately.

Animals↗
PubMed1993

Oncocytes of the thyroid: a neutral red dye uptake study.

Oncocytosis and oncocytomas have attracted attention because on the one hand there have been reports of the favourable prognosis and generally benign nature of oncocytic tumours, while on the other hand, Hurthle cell change in thyroid neoplasms have been reported to be correlated with aggressive biological behaviour and a significantly worse clinical course. Presently, there appear to be two schools of thought on the significance of oncocytosis: (i) a process where there is redifferentiation of cells with involvement in the pathogenesis of cancer; and (ii) a compensatory process for 'functional exhaustion' of the cell. Many aspects of oncocytosis seem unclear. In this study, transmission electron microscopy is used to demonstrate the presence of thyroid oncocytes in the hemithyroidectomy specimen of a patient with focal lymphocytic thyroiditis. Tissue sections from this specimen did not display preferential uptake of neutral red dye, a distinctive characteristic of M-phase cancer cells which was previously reported. The findings seem to suggest that oncocytes are not cells with distinctive proliferative potentials. If that were the case, then oncogenesis or cancer cell progression and oncocytosis may not be convergent processes.

Adenoma↗
PubMed2002

Simple and rapid differentiation of Mycobacterium tuberculosis H37Ra from M. tuberculosis clinical isolates through two cytochemical tests using neutral red and nile blue stains.

The attenuated Mycobacterium tuberculosis strain H37Ra is one of the most commonly used controls for M. tuberculosis identification in the clinical laboratory and is a source of false-positive results for M. tuberculosis as a consequence of cross-contamination. Therefore, the ability to discriminate between H37Ra and real clinical isolates has important public health implications. To date, differentiation of H37Ra from M. tuberculosis clinical isolates is possible only by IS6110 genotyping and spoligotyping. In the 1950s, some authors reported that the virulent strain H37Rv and M. tuberculosis clinical isolates were able to fix basic dyes in their anionic forms, while H37Ra was not. We have studied the different techniques described for M. tuberculosis cytochemical staining and have chosen the best of these, introducing certain modifications in order to increase their discriminative power and reproducibility. We describe cytochemical staining of M. tuberculosis cells with neutral red and Nile blue, which differentiates H37Ra from virulent strains. This method could be used as an easy laboratory tool for distinguishing between H37Ra and real M. tuberculosis clinical isolates.

Bacterial Typing Techniques↗
PubMed2003

Cytotoxicity of liquids and powders of chemically different dental materials evaluated using dimethylthiazol diphenyltetrazolium and neutral red tests.

The purpose of this study was to assess and compare the cytotoxicity of liquid and powder components of chemically different dental materials using 2 basic unspecific cell culture methods. Three chemically cured glass ionomers (Fuji II, Lining cement, and Ketac Silver), 1 light-cured glass ionomer (Fuji II LC), and 2 chemically cured acrylates (Swedon and Super Bond) were tested. The liquids were diluted 1:10 in cell culture medium. The liquids from chemically cured acrylates were further diluted 1:100, 1:1000, and 1:10000. Extracts were made by incubating the powders in cell culture medium for 24 h at 37 degrees C according to the ISO standard 10993-12. The cytotoxicity was assessed in transformed mouse fibroblasts (L-929) using two viability assays, dimethylthiazol diphenyltetrazolium (MTT) and neutral red (NR). Severe cytotoxicity was observed when testing powder extracts of Swedon, Fuji II, and Lining cement, whereas powder extracts of Ketac Silver, Fuji LC, and Super Bond induced slight to non-cytotoxicity. All of the 1:10 liquid dilutions were severely cytotoxic in the MTT assay. In the NR assay, however, four 10% dilutions were severely cytotoxic and 4 moderately cytotoxic. Further dilution of the liquids of the chemically cured acrylates reduced the toxicity, while the Super Bond catalyst was severely cytotoxic even as the 1:100 dilutions.

Animals↗
PubMed2006

Effect of temperature and season on reproduction, neutral red retention and metallothionein responses of earthworms exposed to metals in field soils.

This study investigated the short-term survival, reproduction and physiological (lysosomal membrane stability, metallothionein transcript copy number, body tissue metal concentrations) responses of Lumbricus rubellus exposed to metal contaminated field soils under different laboratory temperatures (10, 15 and 20 degrees C) and physiological responses of earthworms collected from the field in three different seasons (spring, autumn, winter). In the laboratory, metal contaminated soils had significant effects on reproduction (p<0.001), metallothionein-2 (MT-2) expression (p=0.033) and earthworm As (p=0.003), Cd (p=0.001), Pb (p<0.001) and Zn (p<0.001) concentration, but not lysosomal membrane stability and tissue Hg and Cu. No effect of temperature was found for any parameter. Principal component analysis of extractable and tissue metal concentrations indicated PC1 as a measure of metal stress. Both cocoon production (r=-0.75) and MT-2 induction (r=0.41) were correlated with PC1. A correlation was also found between cocoon production and MT-2 expression (r=-0.41). Neutral red retention and MT-2 measurements in worms collected from the field sites in three seasons confirmed the absence of a temperature effect on these responses.

Animals↗
PubMed2005

The neutral red lysosomal retention assay and Comet assay on haemolymph cells from mussels (Mytilus edulis) and fish (Symphodus melops) exposed to styrene.

Despite the extensive transport of chemicals at sea, there is current lack of knowledge of the fate and effects of many of them on the marine biota. The current regulation that follows the GESAMP-MARPOL classification is mainly based on ecotoxicity assessment from fresh water based studies. Repetitive spills in marine coastal environment from tanker ship loaded with several thousand tonnes of chemicals raised concern about whether the existing freshwater data location can be used to predict the behaviour and the environmental effects of contaminants in marine surroundings. There is a general lack of information of the fate of chemicals at sea. A deviating pattern in marine environment from that in freshwater may have significant consequences for the counteracting actions taken to fight the spill, on staff working on the site of spill as well as on marine life present in the vicinity of the accident. In the present article, an environmental effect study of styrene was conducted as part of the ECOPEL program. We report some biological effects of styrene in laboratory-exposed marine organisms. Styrene was continuously supplied at a nominal concentration of 2mg L(-1) over 7 days to both mussels (Mytilus edulis) and fish (Symphodus mellops). At the end of this period, DNA damage was assessed by the Comet assay performed on blood (fish) and haemolymph (mussel) cells. In mussels, the lysosomal membrane stability was additionally assessed by the neutral red retention time assay (NRRT). Significant biological responses were observed over the studied period in both organisms with these two tests. Hence, the results favour the use of a biomarker-based approach to assess the health conditions in case of spill.

Animals↗
PubMed1987

An assay for leukoagglutinating lectins using suspension cultured mouse lymphoma cells (BW5147) stained with neutral red.

A sensitive and rapid assay for leukoagglutinating lectins has been developed. This assay utilizes neutral red-stained mouse lymphoma cells from the suspension cultured cell line BW5147. The agglutination of the stained cells can be monitored visually in a manner similar to that for conventional assays for erythroagglutinating lectins using erythrocytes. The activity of lekoagglutinating lectins that are not capable of agglutinating erythrocytes can be quantified by this assay. The utility of the assay was demonstrated using leukoagglutinating and erythroagglutinating lectins from the seeds of Phaseolus vulgaris and Maackia amurensis.

Animals↗