A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells

Conventionally, a semi-quantitative microscopic nitroblue tetrazolium (NBT) assay is used to determine the production of superoxide anion (O2(-)) in various phagocytic cells. This microscopic assay is conducted by counting the cells containing blue NBT formazan deposits, which are formed by reduction of the membrane permeable, water-soluble, yellow-colored, nitroblue tetrazolium (Y-NBT) by O2(-). However, this assay is semi-quantitative and is prone to observer bias. In the present study, we modified the NBT assay by dissolving the blue formazan particles using 2M potassium hydroxide and dimethylsulfoxide and then measured its absorbance using a microplate reader at 620nm. The absorbance of dissolved NBT increased in proportion to cell number (r = 0.9907), incubation time, and stimulus concentration. To test the usefulness of this modified assay, we compared the abilities of a number of types of phagocytic cells to produce O2(-). The cells examined included murine macrophage cell lines (RAW 264.7 and J774), freshly prepared murine peritoneal macrophages and neutrophils, a human myeloid cell line (PLB-985), and freshly prepared human peripheral blood neutrophils. In addition, we demonstrate that nitric oxide produced by RAW 264.7 cells does not interfere with the modified colorimetric NBT assay. Taken together, our results indicate that the modified colorimetric NBT assay is simple, sensitive, and quantitative, and that it can be used to determine the amounts of intracellular O2(-) produced by phagocytic cells. Thus, this assay is sensitive enough to measure, quantitatively, even the small amounts of O2(-) produced in monocytes and macrophages that are not detectable by the conventional microscopic NBT assay.     

Hypersensitivity in the small intestinal mucosa. V. Induction of cell-mediated immunity to a dietary antigen.

Feeding of a protein antigen to adult mice results in reduced humoral and cell-mediated immune (CMI) responses when that antigen is subsequently presented, and also causes activation of suppressor cells in the gut-associated lymphoid tissues (GALT). We have attempted to abrogate this tolerance to fed antigen by pretreating mice with 100 mg/kg cyclophosphamide before oral immunization and challenge with ovalbumin. Cyclophosphamide-pretreated mice did not develop serum haemagglutinating antibodies, nor systemic CMI (as assessed by skin testing) after ovalbumin feeding. However, evidence that CMI had been induced in the GALT was provided by the significant inhibition of migration and mesenteric lymph node cells from cyclophosphamide-pretreated animals, but not from other control groups. in the presence of ovalbumin. Our previous work on CMI reactions in the small intestine has shown that the cell production rate in the crypts of Lieberkuhn and the intraepithelial lymphocyte count are reliable although indirect measures of mucosal CMI. Cyclophosphamide-pretreated, ovalbumin-immunized animals, which had been fed 0 . 1 mg ovalbumin daily for 10 days before killing, had increased crypt cell mitoses, and increased intraepithelial lymphocyte counts, indicating the presence of mucosal CMI response to ovalbumin. Mechanisms whereby cyclophosphamide pretreatment leads to abrogation of tolerance and induction of mucosal CMI are discussed.     

A rapid and simple MTT-based spectrophotometric assay for determining drug sensitivity in monolayer cultures

Summary A rapid and sensitive spectrophotometric assay for determining viability in monolayer cultured cell lines, with specific applications in normal and drug resistant cell line determinations, is described. The assay involves conversion of the tetrazolium salt MTT by viable proliferating cells to an insoluble product, purple formazan. The chief advantage of this assay is that it requires fewer cells than standard cytotoxicity assays. In addition, it allows for multiple sample concentrations on a single 96-well plate which is then rapidly quantitated using an automated spectrophotometric microplate reader.     

A modified colorimetric assay of macrophage activation for intracellular cytotoxicity against Leishmania parasites

An in vitro method is described which colorimetrically assesses the activation of macrophages for intracellular cytotoxicity against the obligate intracellular parasite Leishmania donovani. The assay system uses a highly purified macrophage population derived from 10-day murine bone marrow cultures. These were infected in vitro as a suspension culture with viable L. donovani amastigotes and then exposed to activating agents. After 48 h the intracellular parasites were released by SDS lysis of the macrophages. Surviving Leishmania organisms were quantitated by their conversion of the chromophore MTT. The sensitivity of this method was comparable with the established method of [3H]dThd incorporation. This assay system has been used to show that there is a dual signal requirement (recombinant interferon-gamma and bacterial endotoxin (LPS] for effective activation of macrophages for leishmanicidal activity.     

测定细胞存活和增殖的MTT方法的建立

MTT方法具有简便,灵敏,稳定可靠,不需要同位素等优点。为了摸索测定细胞存活和增殖的MTT方法的各种最适条件,我们以Raji细胞为对象,比较了十二种不同的MTT甲(月替)溶解缓冲液。实验结果表明,20％SDS-50％DMF效果最好,溶解力强,溶解甲(月替)所需时间短,背景低,OD值高,不会引起培养基中小牛血清的絮状沉淀。其溶解的甲(月替)产物吸收峰在550—600nm之间。测定细胞范围为每孔500—400 000,这一范围能满足生物学和医学研究中多种测定的需要。     

Gender differences in response to the multitest CMI skin test in the general population.

BACKGROUND: Previous studies of gender differences in response to the Multitest CMI skin test have produced conflicting results. OBJECTIVE: To determine whether gender is associated with response to the Multitest CMI skin test. METHODS: Two-hundred ninety-seven adults, aged 18 to 64 years, recruited originally for a study of the immune effects associated with living near a hazardous waste site containing primarily organochlorine pesticides, underwent a skin test using the Multitest CMI skin test. Six of seven antigens were tested: tetanus toxoid, diphtheria toxoid, Candida, Tricophyton, Streptococcus, and Proteus. The tuberculin antigen was excluded. Lymphocyte function was also evaluated in vitro using standardized methods of mitogen stimulation with phytohemaglutinin (PHA), concanavalin A (CON-A), and pokeweed mitogen. RESULTS: The frequency of positive responses to the skin tests was significantly (P < .001) higher among males (80.4%) than among females (55.7%). Males were more likely than females to respond to all six antigens tested (P < .05). The mean diameter of positive skin test measurements for males statistically significantly (P < .05) exceeded female responses for tetanus and diphtheria. Although not statistically significant, male response size exceeded that of females for all other antigens except Trycophyton. Controlling for age, race, smoking, income, and plasma DDE levels did not change these results. Skin test positivity was not associated with mitogen stimulation assay results overall or within gender groups. CONCLUSION: Significant gender differences in response to the Multitest CMI skin test could limit its use as a marker of anergy in general population studies.     

Human cell-mediated immunity to tuberculin as assayed by the agarose micro-droplet leukocyte migration inhibition technique: Comparison with the capillary tube assay

Direct and indirect agarose microdroplet migration inhibition assays were performed with log dilutions (50?5 × 10^?8 μg/ml) of soluble purified protein derivative (PPD) of tuberculin and leukocytes (4 × 10⁵/droplet) from PPD skin test positive or negative individuals. Some tests were run in parallel with the capillary tube method. Inhibition of migration of leukocytes from 9/11 PPD skin test positive donors studied was observed in direct tests when employing PPD doses ranging from 1–50 μg/ml PPD. Inhibition of migration of leukocytes from some PPD skin reactive donors was obtained with as little as 5 × 10^?3? 5 × 10^?7 μg/ml (i.e., 5 nanograms to 0.5 picograms). Some inhibition of leukocyte migration was seen with skin test negative donors (presumably toxicity) with the higher doses of PPD (50 μg/ml), but generally little inhibition was observed with these donors at lower doses. Comparative experiments of the agarose micromethod and the capillary tube technique indicated that the agarose assay was more sensitive in that it could detect reactivity with 2–4 logs lower antigen concentration. Indirect assays using supernatants of cultures of PPD triggered mononuclear cells and indicator granulocytes in agarose droplets demonstrating that a lymphokine (presumably leukocyte inhibitory factor) was being produced and suggested that cell-mediated immune (CMI) reactions were operative. The results indicate the usefulness of this technique in the sensitive detection of CMI against such antigens as soluble PPD. The assay should prove useful in quantitative assessment of cell-mediated reactivity by using a wide range of antigen concentrations and the leukocytes from as little as 2–5 ml of blood.     

A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma-interferon

We describe a simple method by which the insoluble blue formazan dye produced by the reduction of nitro blue tetrazolium can be dissolved without heating using potassium hydroxide and dimethyl sulphoxide. This modification enhances the sensitivity and increases the applications of tests performed using the microELISA method and removes variations caused by uneven cell monolayers. It also allows quantification of NBT reduced by cells adherent to coverslips or in larger wells or Petri dishes, and can be used as a sensitive assay for macrophage activation by gamma-interferon.     

Rapid colorimetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines

A rapid colormetric microtiter assay has been developed to detect cytotoxic lymphokines produced by human lymphocytes activated with lectins or tumor cells. The viability of lymphotoxin-treated target cells was detected using a tetrazolium dye that is reduced to a blue formazan by living but not dead cells. The amount of dye formed was quantitated using a microplate spectrophotometer (ELISA plate reader) and visual observations confirmed the amount of formazan dye produced was directly proportional to the number of viable target cells. The advantages of using this colormetric method are that it requires no washing steps or radioisotopes and its precision and rapidity. Optimal conditions were established using the murine L929 and human ESH-5L cell lines as target cells for detecting lymphotoxins produced by human lymphocytes. The data indicate that the L929 cell line was 10–50-fold more sensitive than the ESH-5L line to the lytic activity of cytotoxins produced by human phytohemagglutinin-P-activated T lymphocytes, or the cytotoxins produced by peripheral blood lymphocytes stimulated with various tumor cell lines. This assay system was also useful in detecting antibodies capable of neutralizing lymphotoxin activity and thus should be a suitable method to aid in the molecular characterization of these lymphokines.     

Rapid colormetric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lymphokines

A rapid colormetric microtiter assay has been developed to detect cytotoxic lymphokines produced by human lymphocytes activated with lectins or tumor cells. The viability of lymphotoxin-treated target cells was detected using a tetrazolium dye that is reduced to a blue formazan by living but not dead cells. The amount of dye formed was quantitated using a microplate spectrophotometer (ELISA plate reader) and visual observations confirmed the amount of formazan dye produced was directly proportional to the number of viable target cells. The advantages of using this colormetric method are that it requires no washing steps or radioisotopes and its precision and rapidity. Optimal conditions were established using the murine L929 and human ESH-5L cell lines as target cells for detecting lymphotoxins produced by human lymphocytes. The data indicate that the L929 cell line was 10–50-fold more sensitive than the ESH-5L line to the lytic activity of cytotoxins produced by human phytohemagglutinin-P-activated T lymphocytes, or the cytotoxins produced by peripheral blood lymphocytes stimulated with various tumor cell lines. This assay system was also useful in detecting antibodies capable of neutralizing lymphotoxin activity and thus should be a suitable method to aid in the molecular characterization of these lymphokines.     

Immune induction of human monocyte plasminogen activator. Characteristics of an assay for cell-mediated immunity

We characterized immunologic induction of monocyte plasminogen activator (PA) to determine whether assay for PA induction reliably detected cell-mediated immunity (CMI). Mononuclear leukocytes (MNL) were incubated in teflon-lined culture tubes for 1-4 days in the presence or absence of phytohemagglutinin-P (PHA), concanavalin A (Con A) or Candida antigen. PA activity of the monocytes in those suspensions was then measured using a micro fibrin plate assay. Monocytes in stimulated MNL had more PA activity than monocytes in unstimulated MNL. Maximal differences between stimulated and unstimulated cells were seen after 2 days of culture. Dose-response studies demonstrated that PA induction occurred at submitogenic concentrations of stimuli. Peak induction was seen using suboptimally mitogenic concentrations of PHA, Con A and Candida antigen. PA induction in response to Candida stimulation corresponded with skin test results. More than 90% of healthy adults tested had positive assays to all stimuli. LPS, in picogram concentrations, induced PA activity in the absence of lymphocytes, but such induction was prevented by polymyxin B. Supernates from activated MNL also induced PA in purified monocytes. This indirect assay of PA induction was less sensitive than direct assay of the MNL. A standard indirect assay for leukocyte inhibitory factor (LIF) was also less sensitive than the direct PA induction assay. The direct PA induction assay is sensitive and convenient and requires small volumes of blood. It may prove valuable in in vitro analysis of cell-mediated immunity in health and disease.     

Detection of grouper mislabelling in the fish market by an immunostick colorimetric ELISA assay

We have applied an immunostick colorimetric enzyme-linked immunosorbent assay (ELISA) assay for the rapid authentication of grouper (Epinephelus marginatus) fillets in the fish market. An indirect ELISA has been assayed by using a monoclonal antibody (3D12) developed previously in another work. In this study, 52 commercial fish fillets samples collected from local markets and supermarkets, which were labelled as grouper, have been analysed by this methodology; only 14 samples were confirmed to be grouper. The simplicity of the procedure and the short time required for the analysis make it suitable for a screening test of a large number of samples and it can be made into a field test for fish processors and inspectors to be used on site.     

A sensitive two-site sandwich enzyme immunoassay for human angiotensin converting enzyme utilizing monoclonal antibodies.

A sensitive enzyme immunoassay was developed for human angiotensin converting enzyme. Monoclonal antibodies specific for two unique converting enzyme epitopes were utilized to develop a two-site sandwich enzyme immunoassay. Alkaline phosphatase conjugated to the detecting antibody hydrolyzes nicotinamide adenine dinucleotide phosphate (NADP) to NAD. Subsequently, NAD is cycled between its reduced and oxidized forms by an alcohol dehydrogenase/diaphorase catalyzed redox cycle. Each cycle converts iodonitrotetrazolium violet to a highly colored formazan which is quantitated. With this assay, as little as 94 pg/ml of native converting enzyme is detectable without interference from either therapeutic or endogenous converting enzyme inhibitors.     

Osteoclast induction from bone marrow cells is due to pro-inflammatory mediators from macrophages exposed to polyethylene particles: a possible mechanism of osteolysis in failed THA

Polyethylene debris from joint replacements may be transported in synovial fluid and be phagocytosed by macrophages. The activation and migration of macrophages may play important roles in osteolysis and implant loosening. Tissues from the bone-implant interface do not always contain wear debris, which may mean that osteolysis may not require direct contact with wear debris. We hypothesized that the release of polyethylene debris from the implants induces macrophage activation in the joint space. Then the activated macrophages release humoral factors, such as inflammatory cytokines, into the joint fluid. These cytokines may be transported to the bone marrow tissues around the implants where they stimulate the differentiation of the bone marrow cells into osteoclasts. Finally, the activated osteoclasts resorb the surrounding bone. To test this hypothesis, macrophages were stimulated by polyethylene particles. The levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were determined by enzyme-linked immunosorbent assay and were increased significantly. To test humoral interaction between macrophages and bone-marrow cells, a co-culture system was used in an in vitro model. With this system, two kinds of cells can be cultured together with humoral contact without the two cell types having to contact each other. We stimulated the macrophages with 5 microm of polyethylene particles and observed whether the bone marrow cells differentiated into the osteoclasts without contact with the macrophages. The numbers of osteoclasts were assessed using tartrate-resistant acid phosphatase (TRAP) staining. The numbers of TRAP-positive cells in the polyethylene particle-stimulated group were higher than in the control group. The ability of the TRAP-positive cells to resorb bone was confirmed by dentine pit formation assay. The results of this study support our hypothesis and suggest that one mechanism of osteolysis in failed joint arthroplasty is the more distant effects of pro-inflammatory cytokine release on osteoclast differentiation and/or activity.     

A rapid colorimetric assay for evaluating Legionella pneumophila growth in macrophages in vitro.

A rapid colorimetric technique for in vitro quantitation of Legionella pneumophila intracellular proliferation in macrophages is described. The assay is based on the electron transport activity of metabolically active L. pneumophila. The yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is cleaved by the mitochondrial activity of viable L. pneumophila, forming a dark formazan derivative with an absorption spectrum different from that of the native compound. The MTT method for measuring intracellular growth of L. pneumophila closely correlated with the CFU assay. The ability of macrophages from the A/J mouse strain to support intracellular growth of L. pneumophila and the ability of desferrioxamine to restrict L. pneumophila intracellular proliferation were confirmed by both methods. The MTT assay offers the advantages of rapidity, simplicity, and cost efficiency over the CFU assay, since it can be performed in the same flat-bottom microtiter plate with measurement in an enzyme-linked immunosorbent assay reader, allowing efficient processing of large numbers of samples.     

Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1 to produce a soluble formazan: a simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents

Activation of the respiratory burst of granulocytes and macrophages by invading microorganisms is a key first line cellular defence against infection. Failure to generate this response leads to persistent life-threatening infection unless appropriate antibiotic treatment is given. The respiratory burst of neutrophils is usually measured spectrophotometrically by following ferricytochrome c reduction, and histologically by using the tetrazolium salt, nitroblue tetrazolium, which is reduced intracellularly to an insoluble formazan. In both assays, reduction is mediated by superoxide generated via NADPH oxidase. Because ferricytochrome c has a high molecular mass and high background absorbance at 550 nm, the assay lacks sensitivity and is not ideally suited to microplate measurement. We have circumvented these limitations by using the cell-impermeable, sulfonated tetrazolium salt, WST-1, which exhibits very low background absorbance and is efficiently reduced by superoxide to a stable water-soluble formazan with high molar absorptivity. This has permitted adaptation of the WST-1 assay to microplate format while retaining sensitivity. Reduction of WST-1 by activated human peripheral blood neutrophils correlated closely with ferricytochrome c reduction across a range of PMA concentrations and with time of activation by PMA and fMLP. Reduction of WST-1 was inhibited by 98% by superoxide dismutase (20 microg/ml) and by 88% by the NADPH oxidase inhibitor, diphenyleneiodinium (10 microM) but was resistant to catalase, azide and the NADH oxidase inhibitor, resiniferatoxin. WST-1 and ferricytochrome c reduction were also compared using xanthine/xanthine oxidase to generate superoxide. Under optimised assay conditions, both WST-1 and ferricytochrome c reduction were directly proportional to added xanthine. WST-1 generated approximately 2-fold greater increase in absorbance than ferricytochrome c at their respective wavelengths, and this translated into increased assay sensitivity. Addition of the intermediate electron acceptor, 1-methoxy phenazine methosulfate, increased the background of the neutrophil assay but did not affect the overall magnitude of the response. We have used the WST-1 assay to assess human neutrophil dysfunction and to compare anti-inflammatory activity.     

Immunosuppression due to chronic ochratoxicosis in broiler chicks

Crystalline ochratoxin A (OA) was added to the feed of broiler chicks at 0.5 ppm and 2.0 ppm, and humoral and cell-mediated immunity (CMI) were studied. CMI was assessed by skin sensitivity testing, graft versus host (GVH) reaction and T lymphocyte count. Humoral immunity was examined by measuring the haemagglutinin (HA) response to sheep RBC (SRBC). In addition, total lymphocyte counts, total serum protein, albumin and globulin were determined and the phagocytic activity of splenic macrophages was measured in the nitroblue tetrazolium test (NBT). The weights of lymphoid organs were also recorded at post-mortem examination of the birds. Highly significant reductions in CMI were indicated by diminished skin sensitivity, GVH reactions and T lymphocyte counts. On the other hand, only the overall HA titres differed significantly between the various treatment groups. Total lymphocyte counts, total serum protein, serum albumin and serum globulin were significantly depressed on the 21st day of intoxication. The number of NBT-positive cells was drastically reduced in both the intoxicated groups compared with controls (P less than 0.05) and the weights of thymus, bursa of Fabricius and spleen of intoxicated birds were significantly reduced. The study illustrated the immunosuppressive effects of ochratoxicosis in broiler chicks.     

An improved spectrofluorometric assay for quantitating yeast phagocytosis in cultures of murine peritoneal macrophages

An accurate means by which to quantitate phagocytosis in murine resident peritoneal macrophages was developed by improving upon existing methods for measuring this important cellular function. Heat-killed Saccharomyces cerevisiae were conjugated to fluorescein isothiocyanate (FITC) and added to macrophage cultures. Following removal of uningested yeast, the macrophages were lysed and the fluorescence associated with the lysates was quantitated. The SEM were rarely +/- 10%. The improved assay was utilized to demonstrate the suppression of yeast phagocytosis by dexamethasone as measured by our radiometric assay. The spectrofluorometric assay produced results similar to those observed when the radiometric assay was employed to determine steroid induced suppression of yeast phagocytosis. However, the improved spectrofluorometric assay is more accurate, reliable, easier to perform, cost and time efficient, and a much safer method for quantitating yeast phagocytosis.     

Assessment of chemokinetic behavior of inflammatory lung macrophages in a linear under-agarose assay

Migration of cells in response to a chemoattractant gradient is influenced by directed migration (chemotaxis) and stimulated random motility (chemokinesis). The present study quantitated the chemokinetic motility of normal and inflammatory lung macrophages by performing the linear under-agarose assay in the presence of uniform concentrations of chemoattractant. Under these conditions, cell motility can be likened to a molecular diffusion process. Mathematical analyses which describe molecular diffusion were then applied, allowing the quantitation of the parameter, mu, the cellular equivalent to the molecular diffusivity constant. Determination of changes in mu as a function of chemoattractant concentration revealed that the chemokinetic motility of alveolar macrophages recovered during the early stages of acute pulmonary inflammation was greater than that of normal alveolar macrophages and macrophages recovered later in the inflammatory response. The correlation of differences in macrophage chemokinesis with macrophage maturation and the relevance of these differences to macrophage accumulation during inflammation are discussed.