High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.     

Cytotoxic effects of FK506 on human renal proximal tubule cells in culture

Summary FK506 has been used as the primary immunosuppressive agent administered after a variety of organ transplants, with less reported nephrotoxicity than that of cyclosporine. This study examined in vitro cytotoxicity of FK506 on normal human renal proximal tubule cells. Cytotoxicity was assessed by neutral red inclusion and trypan blue exclusion; morphology was assessed by light and transmission electron microscopy. Neutral red inclusion decreased to less than 10% of the control after 3 days exposure to 200μg/ml FK506. Forty microgram per milliliter FK506 caused a decrease in neutral red inclusion to 61% of the control on Day 7, with recovery to 86% on Day 12. Similarly, trypan blue exclusion decreased to 66% of the control following 7 days exposure to 40μg/ml FK506, and confluency of the monolayer was reduced to 50% as evidenced by phase contrast microscopy. After a 12-day exposure, treated monolayers became more confluent. On ultrastructural examination, FK506-treated cells exhibited increased cytoplasmic vacuolation and lipid inclusion. These data suggest that FK506 is reversibly and mildly toxic to monolayers of human renal proximal tubule cells and are consistent with clinical reports of reversible nephrotoxicity.     

Tissue Staining by Disazo Dyes, Teratogenicity and the Haemoglobin-Nitrite Sensitivity Reaction

Five disazo dyes related to trypan blue but differing in molecular structure by substitution, replacement or addition of radicals have been tested for their action in increasing the sensitivity of haemoglobin to oxidation by nitrites. None has been found to be as active as trypan blue itself. The activity of these dyes is not related to their ability to cause generalised tissue staining in vivo. Four dyes of known redox potential, neutral red, phenol-indophenol, phenol blue and janus green, have also been tested for their action on the haemoglobin-nitrite sensitivity reaction. Their activity is not related to their redox potential.     

Staining Reactions of some Anionic Disazo Dyes and Histochemical Properties of the Red Impurity in Trypan Blue

Some staining properties of 10 anionic disazo dyes are clarified by comparison with previous chromatographic analysis. Trypan blue contains both blue and red components and the purified blue fraction displays no color shifts in tissue sections. Evans blue, Niagara blue 2B, Niagara sky blue, Niagara sky blue 4B and Niagara sky blue 6B generally resemble trypan blue. Congo red is a metachromatic dye and the only known example among anionic dyes of established purity whose color shows shifts in tissue sections and also in solutions with certain basic compounds. Other red dyes (Congo corinth, trypan red and vital red) are not metachromatic. The red dye impurity of trypan blue selectively stains nuclei which are pycnotic, degenerating or undergoing no further division. This reaction is apparently related to basic protein content. Other reactions of the red fraction of trypan blue (mammalian erythrocytes, blood plasma) are not fully explained on this basis.     

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.     

Studies on the mechanism of natural killer cell-mediated cytotoxicity. VII. functional comparison of human natural killer cytotoxic factors with recombinant lymphotoxin and tumor necrosis factor

The present study was undertaken to evaluate the possible contribution of other cytokines to the lytic activity of NKCF-containing supernatants. We compared some of the functional properties of human NKCF and purified recombinant human rLT and rTNF. It was found that the target cell specificity of rLT was quite different from NKCF in that rLT was neither species specific nor NK specific. Furthermore, antibodies against rLT did not affect the lytic activity of NKCF. These results demonstrate that LT does not significantly contribute to the lytic activity mediated by NKCF. The target specificity of rTNF was found to be related to that of NKCF with the exception of one NK-resistant cell line that was lysed by rTNF in a 20-hr 51Cr-release assay. However, rTNF was not toxic to any of the target cells tested as assessed by trypan blue exclusion in a 20-hr assay unless the targets were labeled with 51Cr. In contrast, NKCF did kill target cells as detected by trypan blue exclusion that were not labeled with 51Cr. Further analysis of this mechanistic difference in the lytic activity of rTNF and NKCF revealed that rTNF in combination with either cycloheximide or mitomycin C but not IFN-gamma could lyse unlabeled U937 target cells. In addition, pretreatment of U937 target cells with nonradioactive Na2CrO4 at concentrations equivalent to that used to 51Cr-labeled cells resulted in their susceptibility to lysis by rTNF as assessed by trypan blue exclusion. These findings suggest that lysis of several susceptible target cells in 20 hr by rTNF requires the presence of additional agents that may be sublethally toxic and/or inhibitory to macromolecular synthesis. Antibody inhibition studies revealed that anti-TNF mediated from partial to complete inhibition of lysis of U937 by unfractionated supernatants containing NKCF. However, fractionation of such supernatants on chromatofocusing columns yielded two distinct peaks of activity eluting in the pH range of 5 to 6 and 7 to 8. Anti-TNF could inhibit the acidic form of NKCF but not the neutral form. It is concluded that NKCF activity is mediated in part by TNF or an antigenically related molecule as well as some other distinct factor(s). The lack of consistent inhibition of NK CMC by anti-TNF suggests that TNF alone is not sufficient to mediate NK activity, or else it is inaccessible to the added antibody.     

Decontamination of aqueous solutions of biological stains.

Aqueous solutions of a number of biological stains were completely decontaminated to the limit of detection using Amberlite resins. Amberlite XAD-16 was the most generally applicable resin but Amberlite XAD-2, Amberlite XAD-4, and Amberlite XAD-7 could be used to decontaminate some solutions. Solutions of acridine orange, alcian blue 8GX, alizarin red S, azure A, azure B, Congo red, cresyl violet acetate, crystal violet, eosin B, erythrosin B, ethidium bromide, Janus green B, methylene blue, neutral red, nigrosin, orcein, propidium iodide, rose Bengal, safranine O, toluidine blue O, and trypan blue could be completely decontaminated to the limit of detection and solutions of eosin Y and Giemsa stain were decontaminated to very low levels (less than 0.02 ppm) using Amberlite XAD-16. Reaction times varied from 10 min to 18 hr. Up to 500 ml of a 100 micrograms/ml solution could be decontaminated per gram of Amberlite XAD-16. Fourteen of the 23 stains tested were found to be mutagenic to Salmonella typhimurium. None of the completely decontaminated solutions were found to be mutagenic.     

Trypan blue dye enters viable cells incubated with the pore-forming toxin HlyII of Bacillus cereus

Trypan blue is a dye that has been widely used for selective staining of dead tissues or cells. Here, we show that the pore-forming toxin HlyII of Bacillus cereus allows trypan blue staining of macrophage cells, despite the cells remaining viable and metabolically active. These findings suggest that the dye enters viable cells through the pores. To our knowledge, this is the first demonstration that trypan blue may enter viable cells. Consequently, the use of trypan blue staining as a marker of vital status should be interpreted with caution. The blue coloration does not necessarily indicate cell lysis, but may rather indicate pore formation in the cell membranes and more generally increased membrane permeability.     

In vitro cytotoxicity of crustacean immunostimulants for lobster (Homarus gammarus) granulocytes demonstrated using the neutral red uptake assay

The neutral red uptake (NRU) cell viability assay was adapted for use with lobster Homarus gammarus (Linnaeus, 1758) granulocytes cultured in vitro. The assay was more sensitive than the conventional trypan blue exclusion assay and facilitated a higher sample throughput than subjective microscope-based assessments of cell viability. The NRU assay was demonstrated to have a linear response from 470 to at least 126000 cells cm(-2). It was used to investigate the acute cytotoxicity of three commercial and two candidate crustacean aquaculture immunostimulants on lobster granulocytes. All five stimulants had a cytotoxic action on the granulocytes and the toxic dose for some of these stimulants was found to be below their commercially prescribed dose. The long term energetic cost of the use of these stimulants and the concomitant potential for a reduction in growth rate of cultured decapod crustaceans, which is fundamental to the success of commercial aquaculture, is identified and discussed.     

ALKALINE RIBONUCLEASE ACTIVITY INCREASE IN RAT KIDNEY CORTEX AND LIVER AFTER TRYPAN BLUE AND OTHER AZO DYES ADMINISTRATION

Acid azo dyes, most of them naphtholdisulfonic acid derivatives, were given intraperitoneally to rats and their effect on "alkaline" ribonuclease activity was studied in total homogenates of kidney cortex and liver. Acid treatment was used to release bound enzyme activity. Several of the dyes, including trypan blue, increased RNase activity in both organs 3 days after administration of single doses, while others, like Evans blue, were inactive. Activity was apparently bound to the sulfonic substitution in the 3, 6 positions in the naphthalene rings, substitutions in the benzidine rings being not critical. All of the active and most of the inactive compounds were taken up by tubule cells of kidney cortex and by reticular and parenchymal cells of liver. While the effect on both liver and kidney was obtained 1 day after trypan blue administration, RNase remained increased for only about 3 days in the first organ, and for at least a month in the second. However, repeated trypan blue doses increased liver enzyme activity for at least 9 days. Serum RNase activity was decreased after trypan blue administration. Ethionine administration together with trypan blue markedly blocked the effect of the dye on liver RNase activity; simultaneously given methionine partially reversed the action of the antimetabolite. This suggests that de novo synthesis of RNase is induced in liver by trypan blue. The action of ethionine on the kidney RNase response to trypan blue was less marked although significant; in view of the possible kidney uptake of the plasma enzyme, interpretation of this finding must be postponed. Results are discussed with reference to the mechanism of the structural specificity of the compounds used, cytological localization of the dyes and their mechanism of action on liver and kidney RNase.     

Microspectrographic analysis of trypan blue-induced fluorescence in oocytes of the Japanese quail

Summary It was shown that the vital dye trypan blue injected subcutaneously is adsorbed on exogenous yolk and stored in oocytes of Japanese quails. The binding sites of the dye could be visualized by fluorescence microscopy. The spectral distribution of the trypan blue-induced fluorescence emitted by yolk granules was analyzed microspectrographically. The analysis revealed that yolk granules exhibit a deep red fluorescence radiation with a maximum intensity at 670 nm, when blue or green excitation light is used. This fluorescence was exclusively induced by the presence of trypan blue, and not by contaminants of the dye. The fluorescence intensity did not decrease during processing of the tissue throughout the different solvents routinely used in light microscopy, especially after fixation in Heidenhain's fluid, nor did it suffer from pronounced fading during irradiation of the tissue. Model experiments showed that the value of the fluorescence emission maximum was concentration-dependent, and that amounts as little as 5×10^–3 mg trypan blue per ml solution containing an excess of yolk as a substrate for the dye, could clearly be detected and measured.It is suggested that a highly diluted solution of trypan blue can be used without teratogenic effects, as a tracer for exogenous yolk uptake and migration into oocytes, and that fluorescence microscopy is a reliable method for its further localization. A detailed account of the procedure is reported.     

Comparison of the Neutral Red and Methylene Blue Assays to Study Cell Growth in Culture

The neutral red and methylene blue in vitro cytotoxicity assays were compared under a variety of conditions using normal human ovarian epithelial cells to determine whether either assay is superior for studying cell growth. The results were standardized against a DNA spectrofluorometric assay. Although the assays were equivalent in reflecting cell number, each has specific advantages: while neutral red discriminates between viable and dead cells, the methylene blue assay is more sensitive and easier to perform.     

Flow cytometric quantitative determination of ingestion by phagocytes needs the distinguishing of overlapping populations of binding and ingesting cells.

BACKGROUND: The use of flow cytometry with fluorescently labeled particles provides the means to examine quantitatively the phagocytotic capacity of an individual phagocyte. This report describes an improved flow cytometric method of analysis for kinetic measurement of phagocytosis of fluorescein isothiocyanate (FITC)-labeled zymosan particles by human leukocytes. METHODS: FITC-labeled zymosan was incubated with leukocyte suspension, and at selected time intervals fluorescence positive neutrophils were divided by phagocytotic gates into three subpopulations: neutrophils that were neither binding nor ingesting particles, neutrophils that were only binding particles (binding cells), and neutrophils that were binding and ingesting particles (ingesting cells). For the distinction between internalized and surface-bound FITC-labeled zymosan, trypan blue (1.2 mg/ml) was used to quench surface-bound fluorescence. RESULTS: The technical challenges related to settings of phagocytotic gates and derivation of phagocytotic equations were presented. From 28 control samples, numerical values of mean fluorescence intensities and percentages of phagocytotic subpopulations inside phagocytotic gates before and after quenching were inserted into phagocytotic equations and corrected phagocytotic parameters were calculated. Calculated parameters were surprisingly constant across individuals. CONCLUSIONS: Essential elements of the present method appeared to be partial quenching of extracellular fluorescence with trypan blue and distinguishing between overlapping populations of binding and ingesting cells. Corrections using derived phagocytotic equations proved necessary for accurate kinetic phagocytotic measurements. Corrections were less necessary when the ingestion process was finished.     

Suppression of cell-mediated tumor cell lysis and complement-induced cytotoxicity by trypan blue.

Different forms of cell-mediated cytotoxicity were suppressed in the presence of trypan blue. The systems affected included lysis of antibody-coated tumor cells by normal and C. parvum-stimulated mouse peritoneal cells and lysis of allogeneic targets by immune effector cells. The inhibition, measured in a 4-hr 51Cr release assay, was reversible and did not occur in the presence of 30% fetal calf serum or albumin. Binding between effector and target cells through Fc receptors was not affected, and lysis of allogeneic cells was inhibited at the lytic step rather than at the binding step. In contrast, lysis of sensitized erythrocytes was not inhibited by trypan blue, suggesting that lysis of these targets may not involve the steps required in tumor cell lysis. Trypan blue blocked the function of antibody before binding to target cells and also suppressed complement-induced cytolysis. Most individual complement components were susceptible to the inhibitory action of trypan blue. These results reveal an affinity of trypan blue for proteins in general that may be responsible for many of its biologic actions.     

Cytotoxicity as measured by trypan blue as a potentially confounding variable in the in vitro alkaline elution/rat hepatocyte assay

Rat hepatocytes treated in vitro with A2RA, an angiotensin II receptor antagonist, displayed increased level of DNA-strand breaks as determined by alkaline elution, without an appreciable increase in cytotoxicity as determined by a trypan blue dye exclusion assay at harvest. The alkaline elution profile appeared to have two components: a rapidly eluting component detected in the first fraction collected (often associated with DNA from dead or dying cells), followed by a more slowly eluting component detected in the subsequent fractions. Further analysis of hepatocytes treated with A2RA by pulsed-field gel electrophoresis and neutral elution revealed significant levels of DNA double-strand breaks. Electron microscopy (EM) showed pronounced damage to mitochondria; although cell blebbing was seen using both EM and light microscopy, the plasma and nuclear membranes appeared intact when examined by EM. Cellular ATP levels decreased precipitously with increasing doses of A2RA, falling to less than 10% of control values at a dose of 0.213 mM A2RA, a concentration showing 100% relative viability by trypan blue at harvest. Thus, whereas in our experience trypan blue dye exclusion accurately reflects cytotoxicity induced by the majority of test agents, in this rather unusual case, trypan blue did not accurately reflect compound-induced cytotoxicity at harvest since there was no concurrent loss of membrane integrity. However, when hepatocytes treated with A2RA were incubated for either 3 h or 20 h in the absence of compound, a sharp, dose-dependent decline in viability was observed using trypan blue dye exclusion. Together with the initial, dose-dependent drop in the alkaline elution curve, these data suggest that the observed DNA double-strand breaks arose as a consequences of endonucleolytic DNA degradation associated with cytotoxicity, rather than by a direct compound-DNA interaction. Since DNA double-strand breaks behave under alkaline denaturing conditions as two single-strand breaks and can therefore produce increases in the alkaline-elution slope values, a necessary criteria for a valid positive result in this assay is that cytotoxicity by trypan blue dye exclusion will not be greater than 30%. Our data, however, indicate that interpretation of the elution assay as a test for genotoxicity can still be confounded by the failure of the trypan blue dye exclusion assay to reflect cytotoxicity in the unusual instance when there is no concurrent, immediate loss of membrane integrity.     

Simultaneous determination of apoptosis and surface antigen expression in tumor adherent cells

Apoptosis is a physiological, gene-directed form of cell death aimed at controlling cell proliferation in several biological conditions. It plays a crucial role in modulating tissue growth during embryonic development, cell turnover in adult life, and it seems to be the most frequent mechanism of tumor cell deletion by chemotherapy. Flow cytometry is a widely-used technique for checking apoptosis, permitting a multiparametric analysis. It is possible to follow the alterations occurring in the nucleus, mitochondria and plasmatic membrane during the different apoptotic stages using probes such as LDS-751, JC-1 or Annexin V. The potential of these probes to identify the early or late stages of apoptosis has been widely investigated in cells growing in suspension. In order to assess apoptosis in adherent cells, we tested a combination of fluorescein diacetate (FDA), a substrate for non specific esterase whose activity decreases during the early phase of apoptosis, and trypan blue in MCF-7 human breast cancer cells. Apoptotic cells showed a decrease in the green fluorescence emitted by fluorescein, the product of FDA hydrolysis, whereas necrotic cells emitted a red fluorescence due to the trypan blue staining. FDA-trypan blue double-staining was used to investigate the different kinetics of apoptosis induced by taxol, camptothecin and UV-B irradiation in MCF-7 cells. This method is rapid and simple, and can be used for monitoring the process of apoptosis from early stages in adherent cells, for the physical separation of apoptotic and live cells, and for immunophenotyping, including Fas expression.     

Utilization of rubidium 86 for humoral cytotoxicity tracing of sarcoma 180]

86Rb has been used as a tracer for Sarcoma 180 ascitic cells in a humoral cytotoxicity system, and compared with the trypan blue dye exclusion method. In as little as five minutes after contact with antiserum and complement, about 90 p. cent of the isotope has gone from the cells, with a significant difference between controls and experimentals. There is a good correlation between 86Rb and trypan blue dye exclusion results. The only pitfall is a relative fragility of cells during the centrifugation stages of the two washings.     

S-antigen in rods and cones of the primate retina: different labeling patterns are revealed with antibodies directed against specific domains in the molecule.

S-antigen (arrestin) is a soluble 48 KD protein of the retinal photoreceptor cells. It has been found to have a function in regulation of the phototransduction cascade. Previous labeling experiments with anti-S-antigen (SAg) antibodies have yielded conflicting reports as to the presence of SAg in cone photoreceptor cells. In the present study we employed five monoclonal anti-SAg antibodies (MAb) directed against different known domains in the SAg molecule. MAb A9C6, D9F2, and C10C10 are directed against sequences in the carboxy half of the SAg molecule. MAb 5C6.47 and F4C1 are directed against the amino terminal. Immunoelectron microscopy was used in the localization of SAg in LR Gold-embedded baboon retinas. Green/red and blue cones were identified with MAb COS-1 and OS-2, respectively. MAb A9C6, D9F2, and C10C10 densely labeled rods and blue cones but not green/red cones. MAb 5C6.47 and F4C1 labeled rods and both blue and green/red cones. It appears that, in the baboon retina, different SAg molecules are present in the blue and green/red cones. Whereas the blue cone SAg shares common antigenic determinants with rods, both in the amino and carboxy terminals, the green/red cone SAg contains different antigenic determinants at the carboxy half of the molecule.     

Selective vital staining of companion cells of potato tuber and parsnip root with neutral red.

When staining the internal phloem region of a potato tuber with the vital stain neutral red, it was observed that files of elongated cells of narrow diameter were heavily stained and were easily distinguishable from the more isodiametric parenchyma cells, many of which did not stain with neutral red. The elongated cells were identified as companion cells by locating the adjacent sieve-tube members through counterstaining with aniline blue and reviewing under violet light. Of a number of other plants surveyed, only parsnip roots possessed companion cells exhibiting a similar slective staining. In other plants both the companion cells and the surrounding parenchyma cells usually stained. Sieve-tube members never accumulated neutral red. It was concluded that the vacuoles of the companion cells of the potato tuber were stained by the ion trap mechanism because of the color of the accumulated stain, the lack of staining when neutral red was applied in an acidic solution, and the complete destaining after soaking in dilute ammonium hydroxide.     

Selective Vital Staining of Companion Cells of Potato Tuber and Parsnip Root with Neutral Red

When staining the internal phloem region of a potato tuber with the vital stain neutral red, it was observed that files of elongated cells of narrow diameter were heavily stained nod were easily distinguishable from the more isodiametric parenchyma cells, many of which did not stain with neutral red. The elongated cells were identified as companion cells by locating the adjacent sieve-tube members through counterstaining with aniline blue and viewing under violet light. Of a numb of other plants surveyed, only parsnip roots possessed companion cells exhibiting a similar selective staining. In other plants both the companion cells and the surrounding parenchyma cells usually stained. Sieve-tube members never accumulated neutral red. It was concluded that the vacuoles of the companion cells of the potato tuber were stained by the ion trap mechanism because of the color of the accumulated stain, the lack of staining when neutral red was applied in an acidic solution, and the complete destaining after waking in dilute ammonium hydroxide.