Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Neutral Red”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,063 records · Page 59Linked to original sources
PubMed1983

Automated colorimetric assay for T cell cytotoxicity.

A colorimetric method has been developed for detecting the lysis of target cells by cytotoxic T lymphocytes (Tc). The method entails incubating Tc cells with thioglycollate-induced macrophage targets and estimating macrophage survival at the end of the assay by staining viable macrophages with the dye neutral red. The method is substantially more sensitive than the 51Cr release assay and can be used to detect alloreactive Tc cells and H-2-restricted Tc cells against viruses, haptens and minor-H antigens. Furthermore, the assay is applicable to limit dilution analysis of Tc cell precursors. The method is cheap, avoids radioactive materials and by measuring optical densities with automated spectrophotometers developed for microELISA systems, results can be obtained 50-100 times faster than with the radioactive procedure.

Animals↗
PubMed1991

A specific and reliable bioassay for the detection of femtomolar levels of human and murine tumor necrosis factors.

A reliable, highly sensitive, cytolytic bioassay for the quantitation of both human and murine tumor necrosis factor (TNF) is described. The assay is 2-180-fold more sensitive than other currently described bio- or immunoassays (limits of detection: 500 fg/ml (29 fmol/l) human TNF-alpha, 200 fg/ml (12 fmol/l) murine TNF-alpha and 130 fg/ml (7 fmol/l) human TNF-beta). The assay, which uses L929-8, a newly isolated subclone of the murine fibroblastoid cell line L929, detects human TNF-alpha approximately 180-fold more sensitively than previously described L929 subclone assays. Maximum sensitivity is obtained by preincubating L929-8 cells at 37 degrees C with 2 micrograms/ml actinomycin D (1-2 h), then culturing with TNF at 40 degrees C for 20 h in medium containing high serum (15% FBS). Relative viable cell content in 96-well microtiter plates is determined colorimetrically by uptake of the non-carcinogenic dye neutral red. Other cytokines have no effect, either alone or in combination with TNF. Cytokines tested were IL-1 through IL-6, GM-CSF, G-CSF, CSF-1, LIF, TGF-beta, NGF, Epo or IFN-gamma, LPS, PGE2, dexamethasone and cyclosporin A, also have no effect, either alone or in combination with TNF. L929-8 cells maintain the above sensitivity to TNF for at least 4 months in continuous culture. Thus, the assay allows rapid, inexpensive, reliable and specific quantitation of rodent and human TNFs. Its very high sensitivity should allow accurate detection of biologically active TNF in biological fluids such as human serum.

Animals↗
PubMed1992

A new approach for realizing the "antioncogram".

On the basis of an organoid culture method which allows organoid reorganization and histotypical growth of human carcinomas under in vitro conditions, we propose in the present study an organoid culture assay (OCA) as "antioncogram", i.e., as in vitro model for testing the drug sensitivity and resistance of individual patients' carcinomas previous to clinical chemotherapy. At the beginning of the assay, specimens of human carcinomas are disaggregated to dense single cell suspensions and dropped on a filter sheet at the air-medium interface. Organoid culture nodules develop within several days to weeks. They are exposed to cytostatics by adding the drugs for 2-3 days to the growth medium below the filter sheet. At the end of the exposure period, the cytotoxic effects are estimated by determining the fraction of viable cells, measured by the uptake of neutral red in relation to the total cell mass. In the present study we could show that this assay actually reflects the different levels of experimentally induced resistance of three human carcinoma strains of an epidermoid hypopharynx carcinoma to the cytostatic drug cisplatin and is obviously suited to predict the response of individual human carcinomas to chemotherapy in a rapid and feasible manner.

Carcinoma, Squamous Cell↗
PubMed1977

Cytogenetic assays of chemical clastogens using mammalian cells in culture.

A protocol is described for cytogenetic assays of chemical mutagens using mammalian cells in vitro. The system employs continuous drug treatment (3 concentrations) for up to 8 h and recovery-cell populations after pulse treatments with a high dose. Both direct fixation (for recording spindle anomalies in anaphase) and colcemid-hypotonic fixation (for reading metaphase chromosome aberrations) are used in order to estimate the effects of an agent as a mitotic poison and as a clastogen respectively. Some DNA intercalating dyes (acridine orange, quinacrine mustard, neutral red) were found to be highly clastogenic whereas others (quinacrine dihydrochloride, 33258 Hoechst) are not.

Cell Line↗
PubMed1991

In vitro cytotoxicity studies with the fish hepatoma cell line, PLHC-1 (Poeciliopsis lucida).

The PLHC-1 fish hepatoma cell line (Poeciliopsis lucida) was used in the neutral red assay to evaluate the acute cytotoxicities of direct-acting (alkylbenzenes, phthalate diesters, and pesticides) and metabolism-mediated (benzo[a]pyrene) toxicants. The sequence of cytotoxic potencies for the alkylbenzenes and phthalate diesters appeared to be a direct function of their hydrophobicity (as described by logarithmic octanol/water partition coefficients). The organochlorine pesticides (alachlor and p,p'-methoxychlor) were more cytotoxic than the organophosphorus pesticides (EPN, diazinon, and malathion). The PLHC-1 cell line apparently maintained sufficient xenobiotic-metabolizing capacity, as the hepatoma cells were able to metabolize benzo[a]pyrene to cytotoxic intermediates. Xenobiotic-metabolizing capacity was temperature dependent, with enzymatic activity increasing as the temperature was increased from 28 to 34 to 37 degrees C, was inducible by Aroclor 1254 (a chemical inducer of cytochrome P450-dependent monooxygenase activity), and was reduced by EPN (an inhibitor of P450 activity).

7-Alkoxycoumarin O-Dealkylase↗
PubMed1987

Purification of adrenal chromaffin cells on Renografin gradients.

Bovine adrenal medullary cells purified on discontinuous gradients of the radiopaque contrast agent Renografin contained about 20% more catecholamine than those prepared by centrifugation on Percoll gradients, a standard method for chromaffin cell purification. Catecholamine recoveries by the two methods were similar, suggesting increased purity in the Renografin preparation. Neutral red staining indicated that the cells prepared on Renografin gradients were approximately 90% chromaffin cells, compared to an average of 75% chromaffin cell purity obtained following centrifugation on self-generated gradients of Percoll. Cells purified on Renografin gradients were equivalent to Percoll-prepared cells by the criteria of maintenance in culture and secretory activity. Thus, density gradient centrifugation in Renografin is a rapid technique for producing adrenal medullary chromaffin cells of high purity and is superior to Percoll gradient centrifugation for this purpose.

Adrenal Glands↗
PubMed1979

Induction of sister chromatid exchanges in the central mudminnow following in vivo exposure to mutagenic agents.

Sister-chromatid exchange (SCE) studies using the central mudminnow, Umbra limi, demonstrated its usefulness as a model in vivo system for the detection of genetically active substances (mutagens and/or carcinogens). 5 days following the injection of 500 micrograms/g of 5-bromodeoxyuridine (BrdU), SCE rates were found to be quite low in the gills (2.0--3.3 SCEs/metaphase), kidneys (2.6--3.4 SCEs/metaphase), and intestines (3.7--4.5 SCEs/metaphase). However, after i.p. exposure to microgram quantities of methyl methanesulfonate (MMS) or cyclophosphamide (CP), large linear dose-dependent increases in SCE rates were observed in all tissues examined. amined. On a microgram/g basis, CP was found to be a 2--4 times more potent inducer of SCE than was MMS. Studies involving the addition of neutral red dye (NR) to the fish's aquarium water revealed that the mudminnow could concentrate the dye in its gill and kidney tissues. This dye was found to cause significant increases in SCE rates at water levels of less than 0.1 ppm. However, the concentration of dye in the tissues did not show a correlation with the SCE rate. Possible explanations for this observation are presented in the text. These findings demonstrate the feasibility of using a fish such as the mudminnow to investigate cytogenetic consequences of aquatic pollution.

Animals↗
PubMed1984

Transplantation of tectal tissue in rats. IV. Maturation of transplants and development of host retinal projection.

We have examined the time course of maturation of embryonic tectal tissue transplanted to the midbrain region of newborn rats and studied the development of the host retinal projection to the grafts. Transplants were examined 2-19 days after transplantation. The morphology of developing transplants was studied using Holmes silver and neutral red stained material. Tectal transplants attained their mature morphology about 17 days after transplantation. The time course of tectal transplant maturation appeared to be similar to that of normal superior colliculus in situ. The development of the host retinal projection into the transplants was examined by injecting the host eyes with horseradish peroxidase (HRP) at various times after transplantation. Retinal fibers anterogradely labeled with HRP were first seen growing into the transplants 3-4 days after transplantation. Ingrowing fibers were always located close to the surface of the transplants. The rate of growth of optic axons into the grafts was estimated to be about 250 to 300 micron per day. Patch-like arborizations of retinal afferents were formed soon after innervation and the mature pattern of optic innervation was established by about two weeks. There was no evidence for an initial transitory phase in which the axons invaded the whole transplant. The development of the host retinal projection preceded morphological maturation of the transplants. The mode of ingrowth of retinal axons into tectal grafts was in many respects similar to the way optic fibers grow into the superior colliculus during normal in situ development. The transplant technique thus provides an opportunity to manipulate and analyze the factors which guide optic fiber growth in intact brains.

Animals↗
PubMed1991

Development of projections of primary afferent fibers from the hindlimb to the gracile nucleus: a WGA-HRP study in the rat.

The projection of primary afferent fibers to the gracile nucleus was studied during development. Injections of wheat germ agglutinin-horseradish peroxidase were made into the hindlimb of fetal, postnatal and adult rats. In most cases the sections were alternately stained for wheat germ agglutinin-horseradish peroxidase including counter stain with Neutral red and for acetylcholinesterase. At embryonic day 17 labelled fibers could be traced to the mid-cervical spinal cord but not further rostrally. At embryonic days E18 and E19 labelled fibers penetrate the rostral pole of the nucleus, which does not happen more caudally. At embryonic day E21 the caudal-most pole of the gracile nucleus still is not penetrated by labelled fibers. From postnatal day 1 onwards labelled fibers are found throughout the entire rostrocaudal extent of the gracile nucleus. These results suggest that primary afferent fibers from the hindlimb first grow to the rostral pole of the gracile nucleus and penetrate the rostral pole immediately upon their arrival. During further development more caudal parts of the gracile nucleus are gradually penetrated in a rostrocaudal fashion by primary afferent fibers of the hindlimb.

Afferent Pathways↗
PubMed1994

Increase of micronucleus frequency in cultured rat hepatocytes treated in vitro with benomyl and pirimiphos-methyl separately and in mixture.

The pesticides benomyl, a benzimidazole fungicide, and pirimiphos-methyl, an organophosphorus insecticide, were tested separately and in combination at a ratio of 6:1, a mixture frequently found in foodstuffs by residual analysis, to determine their possible genotoxic action. The effect was measured by the micronucleus test carried out on cultured rat hepatocytes stimulated to proliferate by epidermal growth factor (EGF). Adult rat hepatocytes were exposed in vitro for 48 h to the substances at increasing non-cytotoxic doses, chosen on the basis of cytotoxicity tests such as LDH and Neutral red assays. Benomyl induced a significant dose-related increase in micronucleus frequency; in contrast, pirimiphos-methyl was not genotoxic at any dose tested. When the hepatocytes were exposed to the two pesticides together at increasing doses, an enhancement in micronucleus frequency similar to that of benomyl alone was found, indicating that at this ratio and non-cytotoxic doses (up to 25 micrograms/ml benomyl + 4.2 micrograms/ml pirimiphos-methyl) no interaction occurs.

Animals↗
PubMed1982

Determination of infectivity of transforming avian sarcoma virus and parotitis virus in fibrin-embedded monolayer cultures.

A sensitive and reproducible technique for titration of transforming avian sarcoma virus, Rous sarcoma virus (RSV), in chick embryo cell monolayers embedded between two layers of a solid fibrin coagulum was developed. The foci of RSV-transformed fibrinolysis-exhibiting cells locally attacked the surrounding fibrin and were identifiable as plaque-like cell-free areas of retraction easily scored against the background of cells stained with neutral red. In comparison to the conventional assay in agar, the assay in fibrin proved more sensitive and less time-consuming. The titration technique in fibrin was also used for plaquing a cytopathogenic virus, parotitis virus, but the titration by conventional plaque assay in agar and the technique in fibrin yielded similar results in this case.

Animals↗
PubMed1990

Use of human monocytes in the evaluation of antiviral drugs: quantitation of HSV-1 cytopathic effects.

An assay for the evaluation of antiviral and immunomodulator potency was developed using pure populations of cultured human monocytes. The assay involved culturing of human monocytes until they were fully susceptible (15-20 days) to lytic infection with HSV-1. When susceptible cells were cultured with recombinant interferon-alpha or a synthetic interferon inducer such as polyinosinic:polycytidylic acid prior to infection, a significant enhancement in resistance to the cytopathic effects of HSV-1 was observed. Likewise, a dose dependent reduction in cell lysis was observed when acyclovir was added immediately after virus infection. Monocyte resistance to HSV-1 was determined by the retention of pinocytic activity as determined by the uptake of neutral red dye. Relative pinocytic activity was quantitated using a simple colorimetric procedure. This antiviral assay can be completed in 48 h; is easy to perform, highly sensitive and reproducible.

Acyclovir↗
PubMed1992

Use of a standardized cell culture assay to assess activities of nucleoside analogs against hepatitis B virus replication.

A cell culture system for the evaluation of compounds which inhibit HBV replication (Korba and Milman, Antiviral Res. 15:217, 1991) has been developed into a standardized assay. Toxicity of test compounds was assessed by the uptake of neutral red dye under culture and treatment conditions which were identical to those used for the antiviral assays. A total of 667 separate cultures of 2.2.15 cells were evaluated for this study. In 86 untreated cell cultures, representing 15 experiments over a 24-month period, the levels of extracellular HBV virion DNA and intracellular HBV DNA forms were found to vary by less than 2.5-fold overall. Virion DNA in serum and intracellular viral DNA replication intermediates [RI] are the two most reliable and commonly followed markers of hepadnavirus replication in patients and experimental animals. In these assays, levels of extracellular HBV virion DNA and intracellular HBV RI were well correlated in 2.2.15 cells. Less correlation was observed between the levels of HBV virion DNA and the 3.2-kb episomal HBV genomes present in the cells. A threshold level of 22-37 intracellular replicating HBV genomes appeared to be required before virions were detected in the culture medium. The activities of several 2'-substituted and 3'-substituted deoxynucleoside analogs against HBV replication were compared using this standardized assay. Dideoxycytosine [ddC] and dideoxyguanosine [ddG] were the most selective 2',3'-dideoxynucleosides against HBV in 2.2.15 cells. Substitution of fluorine at the 2' position abolished the antiviral activity of ddC, but enhanced the selective antiviral activities of dideoxythymidine and dideoxyuracil. Several 2'-fluorinated pyrimidine arabinosyl furanosides, reported to be potent (but toxic) inhibitors of hepadnaviruses in vivo, demonstrated relatively low selective antiviral activities in 2.2.15 cells. The current data base allows for validation of any given set of test evaluations through statistical analysis of both the positive and the negative treatment controls present in each experiment; thus, relevant comparisons of the selectivity of anti-HBV activities for different compounds examined in future experiments can be made.

Antiviral Agents↗
PubMed1995

SDZ 35-682, a new picornavirus capsid-binding agent with potent antiviral activity.

SDZ 35-682 is a potent and selective inhibitor of the replication of members of the picornavirus group. It inhibits several rhinovirus serotypes and echovirus 9 at concentrations as low as 0.1 micrograms/ml, without exerting any effect on cell proliferation up to 30 micrograms/ml. As observed with other capsid-binding antipicornavirus compounds, there is a wide variation in sensitivity of the different serotypes within the rhinovirus group. The point of interference of SDZ 35-682 in a single cycle of virus growth is an early event taking place before 2 or 3 h of echo- or rhinovirus replication, respectively. By incorporation of neutral red into the viral capsid and measurement of acquisition of photoresistance it is shown that uncoating of echovirus 9 is inhibited by SDZ 35-682. In addition, efficiency of adsorption of echovirus 9 is reduced by SDZ 35-682. To demonstrate that SDZ 35-682, like other uncoating inhibitors of picornaviruses, binds to the hydrophobic pocket beneath the canyon floor co-crystallization with HRV 14 was performed. Considerable conformational changes occur in VP1 in the HRV 14/SDZ 35-682 complex. SDZ 35-682 is 19 A long from end to end and thus fills the entire hydrophobic pocket including its innermost end; it is less flexible than other long antiviral agents. It has been suggested that compounds filling the entire hydrophobic pocket will affect the uncoating process of the virion. Thus, inhibition of viral uncoating, as demonstrated with echovirus 9, probably is the predominant mode of action of SDZ 35-682.

Animals↗
PubMed1995

Anti-human cytomegalovirus activity and toxicity of sulfonated anthraquinones and anthraquinone derivatives.

Sulfonated anthraquinones and other anthraquinone derivatives were evaluated for anti-human cytomegalovirus (HCMV) activity, cytotoxicity and genotoxicity. Acid blues 40 and 129, acid black 48, alizarin violet R and reactive blue 2 were the most active compounds having selective indices of greater than 30 and EC50 values of 4-30 microM. When tested against a clinical isolate, the 4 compounds were 2- to 5-fold less active. The antiviral activity was distinctly separate from the virucidal activity (> 1000 microM). The compounds were weakly toxic to either log phase or stationary cells in most of the following cytotoxicity assays: neutral red uptake assay, lactic acid dehydrogenase assay, trypan blue exclusion assay and radiolabeled macromolecular precursor uptake assays. Using a genotoxicity assay, the comet assay, only reactive blue 2 and acid black 48 were found to cause DNA strand breakage. This occurred at concentrations of 30 and 170 microM, respectively. These results suggest that these compounds could be a prototype for synthesizing even more effective HCMV-inhibitory anthraquinone derivatives.

Anthraquinones↗
PubMed1994

Development of antibiotic-resistant strains for the enumeration of foodborne pathogenic bacteria in stored foods.

Strains of Aeromonas spp., Salmonella enteritidis phage type 4, Salmonella typhimurium, verotoxigenic Escherichia coli O157:H7 (VTEC) and Yersinia enterocolitica resistant to streptomycin, nalidixic acid and a combination of both antibiotics were selected. When compared with the parent strains, most of the antibiotic-resistant strains had slightly slower growth rates at their optimum incubation temperature but the difference was reduced progressively when the temperature was lowered. Some antibiotic-resistant strains had considerably slower growth rates in the presence of the relevant antibiotic and these were not used further. Several agar and impedance media with added streptomycin and nalidixic acid were assessed for the enumeration of the antibiotic-resistant strains in artificially contaminated stored foods. Differential/selective media were required to enumerate low numbers of antibiotic-resistant strains in certain foods. The following agar and impedance media were selected: Aeromonas Agar (Ryan) for Aeromonas spp., Xylose Lysine Agar and Lysine Iron Cysteine Neutral Red Medium for Salmonella, Eosin Methylene Blue Agar and Coliform Medium for VTEC, and Yersinia Selective Agar without selective agents for Yersinia enterocolitica. The agar and impedance media have been used successfully to enumerate antibiotic-resistant strains inoculated into foods and stored at different temperatures.

Aeromonas↗
PubMed1992

Cellular alterations produced by the experimental increase in intracellular calcium and the nature of protective effects from pretreatment with nimodipine.

The immortalized septal cell line, SN56 B5 G4, generated by the fusion of mouse septal area cells and neuroblastoma cells, was used to determine if nimodipine, an antagonist of voltage sensitive calcium 'L' channels, might act in a neuroprotective fashion when intracellular calcium levels were raised by incubation in ouabain and monensin. Fluorescent indicator dyes and the automated spectrofluorometer, the CytoFluor 2300, were used to analyze specific cellular targets and functions affected by ouabain and monensin and possible protection by prior incubation with nimodipine. Ouabain and monensin were used together to create a time- and dose-dependent toxic episode. Increases in the emission intensity of Fluo3-AM demonstrated that the concentration of intracellular calcium was monotonically increased by increasing levels of ouabain-monensin. The calcein-AM fluorescent probe indicated that there were no changes in plasma membrane permeability during the toxic episode. Lysosomal integrity decreased as indicated by decreases in neutral red retention. The concentration of free radicals increased as shown by the increase in emission intensity of 2',7'-dichlorfluorescein. Nimodipine pretreatment of the cells incubated with ouabain and monensin resulted in apparent protection of lysosomes and a reduction in the level of free radicals. While nimodipine, by itself, produced a small decrease in intracellular calcium, it actually augmented the ouabain-monensin induced increase in intracellular calcium. The data suggest that in immortalized septal cells, (a) nimodipine offers protection to certain of the responses induced by ouabain-monensin, (b) the protection offered by nimodipine may be independent of antagonism of voltage sensitive calcium channels, and (c) that the protective changes can occur at the same time that intracellular calcium is increasing. These latter observations question the hypothesis that the protection against cell death and dysfunction offered by nimodipine is due solely to maintaining calcium homeostasis.

Animals↗
PubMed1993

Evaluation of biocompatibility of various ceramic powders with human fibroblasts in vitro.

Cell reaction to powders of ceramics was studied in vitro. Cultured human fibroblasts were exposed to different types of ceramic powders: zirconia (ZP), alumina (A), tricalcium phosphate (TCP) and hydroxyapatite (HA), at various concentrations. The cell viability at the different exposure times was measured by the colony formation (expressed as colony forming efficiency, CFE), neutral red uptake (NR) and colorimetric tetrazolium (MTT) reduction. Alumina and hydroxyapatite showed no cytotoxic effects at studied doses (1-500 mug/ml) while zirconia and tricalcium phosphate inhibited cell viability, with 50% of CFE reduction at the concentration of about 50 mug/ml. In order to study the cytotoxic mechanism of zirconia powder, two further experiments were included, viz. the cellular response to the sintered zirconia ceramic powders (CZP) which were obtained by crushing the sintered ceramic material; and the measurement of the degradation of zirconia ceramic plate in the different solutions, i.e., either in saline or in 0.02 M lactic acid (pH 2.72). Similar cell reactions were obtained for the CZP and ZP by using MTT and NR assays. Slow releases of ions from zirconia ceramic plate, yttrium in both solutions and zirconium and yttrium in lactic acid, were detected.

Aluminum↗