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Simultaneous evaluation of viability and acrosome integrity of mouse spermatozoa using light microscopy.

Determination of the percentage of live cells with intact acrosomes and no morphologic aberrations could be a practical index of semen quality. We applied viability and acrosome staining techniques, originally described for bull, boar and rabbit sperm, to mouse spermatozoa. The viability stain was either trypan blue or Congo red. The stain was precipitated by neutral red in the fixative. The acrosome was stained by Giemsa. Sperm morphology, including cytoplasmic droplets, could be evaluated as well. The staining method described here is a useful routine tool for simultaneous evaluation of the plasma membrane integrity of different sperm subdomains, the status of the acrosome, and cellular morphology.

Acrosome↗

Practical applications of neuronal tissue culture in in vitro toxicology.

1. Primary chick embryo forebrain neurones are relatively easy to culture and are quite resilient to treatment manipulation. These characteristics have allowed application in a surprisingly diverse number of areas. 2. These cultures have been used to investigate the anticholinesterase potencies of many organophosphate (OP) nerve agents and insecticides. 3. These cultures have been used to quantitate levels of OP in 'spiked' unknowns and in OP-contaminated soil samples. 4. These cells have also been used to test a variety of compounds using the MTT and neutral red cytotoxicity assays.

Animals↗

Mouse peritoneal cells activated with a combination of indomethacin, poly I:C and Syncumar inhibit the take of Lewis lung carcinoma in adoptive transfer assay.

Effect of peritoneal cells (PC) from mice treated with a combination of drugs (indomethacin, poly I:C and Syncumar) on the take of Lewis lung (LL) carcinoma was studied in Winn-type adoptive transfer experiments. Transfer of PC from mice given a single intraperitoneal injection of polyinosinic-polycytidylic acid (poly I:C) or indomethacine or Syncumar (100 micrograms of each) per se did not suppress the take of Lewis lung carcinoma in the recipient mice. PC obtained from mice treated with a combination of indomethacin and poly I:C or poly I:C and Syncumar also failed to inhibit the take of the tumour. In contrast, PC collected from mice after a combined treatment with the three drugs (indomethacin + poly I:C + Syncumar) resulted in a 30-60% decrease in tumour take depending on the tumour cell/PC ratio. This effect could not be observed when a single intraperitoneal dose of cyclophosphamide was administered three days before starting of the combined treatment of the donor mice. The effector cells contributing to the tumour inhibitory effect proved to be nonadherent cells, probably large granular lymphocytes (LGL), as their suppressive effect was abrogated after treatment with the lysosomotrop vital dye neutral red.

Acenocoumarol↗

Assessment of cytotoxicity using electric cell-substrate impedance sensing: concentration and time response function approach.

This paper describes a simple and convenient method to measure the concentration and time response function f (C,t) of cells exposed to a toxicant by electric cell-substrate impedance sensing. Attachment and spreading of fibroblastic V79 cells cultured on small gold electrodes precoated with fibronectin were detected as electrical resistance changes. With this method, chemical cytotoxicity was easily screened by observing the response function of attached cells in the presence of inhibitor. The cytotoxicities of three test models, cadmium chloride, sodium arsenate, and benzalkonium chloride, were quantified by measuring the percentage inhibition as a function of the inhibitor concentration. The half-inhibition concentration, the required concentration to achieve 50% inhibition, derived from the response function agreed well with the results obtained using the standard neutral red assay.

Animals↗

[Polarity of cell permeability and its control by phytochrome].

Growing chloronema and rhizoid cells of the intact protonema of Funaria hygrometrica absorb luminescent stains selectively. The cation stains--acridine orange and neutral red, at pH 5.0 are absorbed by growing tips of the apical cells. The anion stain (uranine) is absorbed by the basal end of the apical cells and by intercalary cells of the protonema filaments. Red light inactivates the cell permeability to the cation and anion stains. Far-red light partly reversed the action of the red light solely on the initial stages of the irradiation. During the red light induced branching of apical cells the cation stains enter the cells through a new originated growing point.

Acridines↗

[Dependence of frog epithelial cell and neuron survival time on the concentration of certain substances].

The frog cornea epitelial cells, nervous cells of spinal ganglia and of urinary bladder have been found to survive in vitro, in the presence of subtoxical concentrations of chloral hydrate, urea, ethanol or methanol added to the Ringer solution, longer than control preparations. The above drugs added both in lower and higher concentrations lead to cell damages, thus reducing the cell survival time, which was tested by the capacity to deposit granules of a vital dye - neutral red.

Animals↗

A direct plaque assay for hog cholera virus.

Direct plaque formation with representative strains of hog cholera virus (HCV) has been obtained using several pig kidney cell lines under agar overlay. HCV-infected cells appear as hazy plaques when viewed against an indirect light source, and as white plaques after neutral red staining. HCV assay by direct plaque procedure is rapid and convenient and gives infectivity titres identical to the fluorescent focus assay technique.

Cell Line↗

Development, hatching, and infectivity of Echinostoma caproni (Trematoda) eggs, and histologic and histochemical observations on the miracidia.

Light microscopy studies were done on the eggs and miracidia of the intestinal trematode Echinostoma caproni. Eggs were obtained by homogenizing adult worms derived from ICR mice in artificial spring water (ASW). To study egg development and to obtain miracidia, eggs were incubated in ASW in either a light or a dark environment for 10 days at 28 degrees C. Egg development was characterized and staged at 2-day intervals from day 0 to day 10 postembryonation. No significant difference in development was seen in eggs maintained under conditions of light or darkness. Eggs maintained in darkness for 10 days and exposed to incandescent light produced a large synchronous hatch of miracidia within 3 h of exposure to light. As expected, miracidia used to expose Biomphalaria glabrata snails produced patent infections at 5-7 weeks postinfection (p.i.). Embryonated eggs aged from 0 to 10 days that had been fed to B. glabrata snails also produced infections within 4 weeks of infection. Miracidial longevity studies showed that 50 newly hatched miracidia maintained at either 4 degrees, 12 degrees, 22 degrees, or 38 degrees C lived for 6, 28, 14, and 5 h, respectively. Histology studies were done on whole miracidia stained in 0.01% neutral red and in Schneider's acetocarmine. Histochemistry studies of miracidia stained with alcian blue (pH 2.5) showed the presence of acid mucopolysaccharides in the epidermal plates; miracidia treated with periodic acid-Schiff plus 0.5% malt diastase showed the presence of glycogen in the body and in the epidermal/subepidermal region. Miracidia stained with Lillie's Oil Red O showed the presence of sparse neutral fat droplets in the body.

Animals↗

Establishment of immortalized cell lines from mouse peritoneal macrophages following transformation with SV40 early region DNA deleted at the origin of replication.

Two murine peritoneal macrophage cell lines have been isolated by transforming primary cells with simian virus 40 (SV40) origin-deleted DNA. These lines have been maintained in continuous culture for over 8 months and have been shown to express macrophage-specific properties throughout this time. The cell lines are F4/80 positive; express Fc receptors; will phagocytose immunoglobulin-coated red cells and latex beads; stain with neutral red; and have non-specific esterase and plasminogen activator activities. Lysozyme, collagenase, prostaglandin E2, acid phosphatase and 5'-nucleotidase activities have also been detected and quantified.

Animals↗

Development of a respiratory burst assay using zebrafish kidneys and embryos.

The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including the respiratory burst of phagocytes. Respiratory burst can be used as a reliable measure of the immune response of a host, and numerous assays have been developed to measure this response in a variety of mammal and fish species. Phagocytes, like granulocytes and macrophages, that are derived from different tissues, or grown in cell culture, have been employed in a range of assay formats employing a variety of detection methods. The small size of the zebrafish has prevented the large-scale extraction of these cells for respiratory burst assays in the zebrafish. In this work, we describe a respiratory burst assay developed for the zebrafish using intact kidneys and embryos as sources of phagocytes. Phorbol myristate acetate (PMA)-inducible reactive oxygen species (ROS) were detected following the oxidation of a non-fluorescent dye 2',7'-dihydrodichlorofluorescein diacetate (H2DCFDA) to dichlorofluorescein (DCF), a fluorescent product. Embryos from 1 day post-fertilization until 5 days post-fertilization (dpf) were employed in this assay. Abrogation of H2DCFDA oxidation by the protein kinase C (PKC) inhibitor bisindolylmaleimide I (BisI) indicated a reduction in the respiratory burst. Fluorescence from the PMA-induced respiratory burst in kidneys and embryos was significantly elevated above DMSO-treated controls, while preincubation with BisI inhibited the increase in fluorescence. Colocalization of cell-associated chloromethyl-dihydrodichlorofluorescein diacetate (CM-H2DCFDA) with the phagocyte-selective dye neutral red is consistent with the observation that macrophages and granulocytes are the ROS-producing cells in the zebrafish.

Animals↗

Specific accumulation of GFP in a non-acidic vacuolar compartment via a C-terminal propeptide-mediated sorting pathway.

The green fluorescent protein (GFP) from Aequorea victoria can be detected in living plant cells after transient transformation of protoplasts. Expression of the GFP can be used to monitor protein trafficking in a mixed cell population and also to study the different function and importance of organelles in different cell types. We developed a vacuolar form of GFP that was obtained by replacing the C-terminal endoplasmic reticulum (ER)-retention motif of mGFP5-ER by the vacuolar targeting peptide of tobacco chitinase A. The vacuolar GFP was transported and accumulated in the vacuole as expected. However, we found two patterns of GFP accumulation after prolonged incubation (18-24 h) depending on the cell type. Most chloroplast-rich protoplasts had a fluorescent large central vacuole. In contrast, most chloroplast-poor protoplasts accumulated the GFP in one smaller vacuole but not in the large central vacuole, which was visible under a light microscope in the same cell. This differential accumulation reflected the existence of two different vacuolar compartments as described recently by immunolocalization of several vacuolar markers. We were able to characterize the vacuolar compartment to which GFP is specifically targeted as non-acidic, since it did not accumulate neutral red while acidic vacuoles did not accumulate GFP.

Androstadienes↗

Identification of beta-cells in dissociated rat pancreatic cell suspensions.

beta-Cells may be demonstrated in pancreas sections as well as in monolayer cultures. Until now, however, it has been impossible to differentiate quickly among individual cell types in freshly dissociated cell suspensions prepared for pancreatic monolayer cultures. Rapid identification of endocrine cells within the total cell population can be achieved by means of vital staining with neutral red. Moreover, the direct observation of unfixed and unstained cell suspensions under dark-field illumination allows an immediate identification of granulated beta-cells.

Animals↗

Affinity chromatography of viroid RNAs.

The application of a structure- and GC-specific affinity adsorbent for the purification of viroid RNAs is described. Cross-linked bisacrylamide gels to which the intercalating dye phenyl neutral red is covalently attached [2] constitute a powerful tool for the rapid, quantitative, and selective isolation of viroid as well as viroid complementary RNAs from crude, plant polysaccharides containing nucleic acid fractions.

Aminoacridines↗

Molecular characterization of anterior-like cells in Dictyostelium discoideum.

Prestalk cells are found in the anterior 15% of slugs of Dictyostelium discoideum and prespore cells are found in the remaining posterior portions. Neutral red staining as well as immunostaining with antisera prepared against spores or a spore-coat protein has indicated that there are cells in the posterior region which have properties similar to prestalk cells. This cell population, called anterior-like cells, has been isolated by density separation of posterior slug fractions. This anterior-like cell preparation contained approximately 80% neutral red-stained cells, none of which carried a surface antigen specific to prespore cells (MUD-1 antigen). These cells also lacked antigens recognized by antisera to the spore-coat proteins or by a prespore-specific monoclonal antibody isolated for this study (mLJ1). Anterior-like cells were found to modify the lysosomal enzymes acid phosphatase and alpha-mannosidase in a manner found specifically in prestalk cells. They also synthesized two prestalk-specific proteins and contain two prestalk-specific mRNA transcripts. Anterior-like cells also failed to express a large number of prespore-specific characteristics. Although very similar to prestalk cells, anterior-like cells differ from them in that they express a prespore-specific protein PSP59 and the levels of the prestalk-specific mRNA transcripts and one of the prestalk-specific proteins ST430 appear to be lower.

Acid Phosphatase↗

[Effect of the fungal metabolites of Aspergillus flavus and A. niger on the tissue sorptive capacity of the large intestine].

Effects of metabolites of the tiny fungi of Aspergillus flavus and A. niger on the ability of mucuos membrane cells of the large intestine to absorb and exterminate certain dyes has been studied experimentally on guinea-pigs. It has been shown that under these influences, a violation of the cell and tissue metabolism in the large intestine occurs, namely, the absorbtion of the neutral red increased by 1.7 and 1.4 times, respectively, and the processes of extermination of the cells were inhibited by 1.5--1.6 times.

Animals↗

[Cytotoxicity of DMSO for MRC5, Chang liver and CV1 cells evaluated in vitro by LK, MTT and NR assays].

Evaluation of chemicals cytotoxicity plays fundamental role in many in vitro investigations. The way of assessment of cytotoxicity depend on aim of study, characteristic of used cells and mode of action of investigated chemicals. The principal aspect of these investigations is validation of used method. In this paper validation of three different cytotoxicity assays is presented: total cell number measurement (LK), microplate assay measured mitochondrial dehydrogenase activity (MTT) and colorimetric assay measured ability of live cell to uptake neutral red (NR). The investigation was performed on different cells (MRC5, CV1 i Chang Liver) with DMSO as reference agent.

Animals↗

[The modification of Bacillus intermedius ribonuclease by urea derivatives].

The results of the "Bacillus intermedius" RNAase modification by urea derivatives are presented. The modifiers synthesized for that purpose were the following: N-(4-chlorbenzoyl)-N'-benzolsulfonyl urea (M1) and N-(4-chlorbenzoyl)carbomoyl-epsilon-aminocapronic acid (M2). It was shown that RNAase modified by M2 stimulated the cell general metabolism by the test of the cell absorption of neutral red and had a more marked ability to disrupt RNA of the cell plasmatic membranes in comparison to that of the native enzyme.

Amnion↗

Evaluation of PREDISAFE, a cell kit for predicting eye irritancy of cosmetic raw materials and formulations.

A cell kit named PREDISAFE based on the use of confluent rabbit fibroblastic cells has been designed to predict eye irritancy of cosmetic raw materials and formulations. The kit can be stored for a few days and/or shipped at room temperature. Cytotoxicity was estimated after 1 min or 15 min contact with test compounds using the neutral red release assay. For the 84 products tested, IC50 values gave intervals similar to classes defined from the Draize test, i.e., mild, moderate, severe and extreme irritancy.

Animals↗