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At least 739 records · Page 41Linked to original sources
PubMed2005

Application of MTT reduction assay to evaluate equine sperm viability.

The assay of MTT reduction depends on the ability of metabolically active cells to reduce the tetrazolium salt (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) to formazan. This study was conducted to examine and validate of a simple and less costly MTT test in determining equine sperm viability and compare the efficiency of this test with a flow cytometer. Fresh ejaculates from 11 stallions (warm blood) were included in this study. Semen was diluted to 100 million cells/ml in a Hepes 0.1% BSA. The rates of MTT reduction were measured in microtiter plates after incubation for 1 and 4h at 37 degrees C using spectrophotometer (MS2 Reader) at wavelength 550 nm. Simultaneously split samples of the same semen were tested, using a flow cytometer for mitochondrial activity, sperm viability, and acrosomal integrity using Rhodamine 123, SYBR-14 and LysoTracker Green DNA-26, respectively. The results revealed a strong correlation (P < 0.001) between the results of MTT test at 1 and 4 h of incubation time and the result of mitochondrial activity (r = 0.978, 0.977), sperm viability (r = 0.954, 0.977) and acrosomal integrity (r = 0.867, 0.886). In conclusion, the MTT test was found to be a reliable method in evaluating semen viability and can be used successfully, especially in routine analysis, where practical aspects such as time, costs and practicability are important.

Acrosome↗
PubMed2005

Effects of natural prenylated flavones in the phenotypical ER (+) MCF-7 and ER (-) MDA-MB-231 human breast cancer cells.

The effect of seven natural prenylated flavones in DNA synthesis of two human breast cancer cell lines, the estrogen-dependent ER (+) MCF-7 and the estrogen-independent ER (-) MDA-MB-231 cells, was evaluated. Flavones with an isopentenyl group at C-8 and a ring linking C-3 and C-2' presented a biphasic effect in DNA synthesis of ER (+) MCF-7 and displayed a stimulation at low concentrations (0.02-0.78 microM) whilst at higher concentrations (> 3.12 microM) inhibition was observed. No stimulation was observed in ER (-) MDA-MB-231. In contrast, all the flavones exhibited an antiproliferative effect in both ER (-) and ER (+). Curiously, the inhibition of DNA synthesis was accompanied by a high capacity of these cells to reduce MTT, which was concurrent with the appearance of an intense intracytoplasmic vacuolization. The accumulation of the formazan product in these vacuoles could justify the enhancements of MTT reduction. The characterization of these vacuoles with the autophagic marker monodansylcadaverine (MDC) is consistent with autophagic vacuoles, which led to the suggestion that these flavones could induce autophagy in both ER (+) and ER (-) breast cancer cell lines.

Breast Neoplasms↗
PubMedDate not supplied

Adaption of neutral protease activity for islet isolation from the long-term two-layer method-stored pig pancreas.

BACKGROUND: Islet release from the pancreas is mediated by both collagenase and neutral protease (NP), a critical effector of islet integrity. To prove the hypothesis that adjustment of NP reduces islet damage after prolonged ischemia, adult pig pancreata were digested after 7-hour preservation by the two-layer method (TLM) using a 2-component enzyme blend consisting of collagenase NB-8 and NP. METHODS: After intraductal University of Wisconsin (UW) flush resected pancreata were distended with 4.4 PZ-U/g of UW-dissolved Serva collagenase either before (TLM-preloaded, n = 7) or after (TLM-postloaded, n = 10) cold storage, or for immediate processing (n = 6). NP was adjusted after preliminary experiments to respectively 1.1, 0.2, or 0.8 DMC-U/g for unstored, TLM-preloaded, or postloaded organs. RESULTS: Purified islet yield decreased from 3670 +/- 730 islet equivalents (IEQ)/g in unstored pancreata to 1800 +/- 180 and 2080 +/- 290 IEQ/g in TLM-preloaded or postloaded organs, respectively (P < .05). Although purity was always >90%, IEQ recovery was significantly decreased in TLM-preloaded pancreata. Quality control revealed consistently high viability as determined using trypan-blue exclusion (>95%) or formazan production. Compared with unstored organs (2.47 +/- 0.36; P < .05), glucose stimulation index was reduced in TLM-preloaded (1.48 +/- 0.15) and TLM-postloaded pancreata (1.81 +/- 0.20). Normoglycemia in diabetic nude mice transplanted with islets from TLM-preloaded pancreata was transient in contrast to sustained function in the other groups. CONCLUSIONS: Significant amounts of viable pig islets can be isolated after prolonged TLM preservation by reducing NP activity. Nevertheless, early enzyme administration prior to long-term storage deteriorates islet graft function.

Adenosine↗
PubMedDate not supplied

Long-term preservation of the pig pancreas by a one-layer method for successful islet isolation.

BACKGROUND: Pancreas preservation by two-layer method (TLM) was recently established for clinical islet transplantation. The extensive use of TLM would require enormous efforts to solve logistical and technical problems. Omitting University of Wisconsin solution (UW) as second layer would facilitate the regular application of oxygenated perfluorocarbon; (PFC). To clarify whether long-term pancreas preservation is feasible by this simplified procedure, pancreases from retired breeder pigs were subjected to 7-hour preservation utilizing PFC alone in a one-layer method (OLM, n = 8) or in combination with UW (TLM, n = 10). METHODS: Resected pancreata were intraductally flushed with cold UW. Subsequently, pancreata were promptly processed (n = 6) as previously described or stored by TLM or OLM. RESULTS: Compared to unstored (429200 +/- 86700 IEQ) and OLM-stored pancreases (338600 +/- 42100 IEQ), (P = ns vs unstored) postpurification islet yield decreased after TLM storage (238000 +/- 26600 IEQ, P < .05). No significant differences were found regarding purity (>90%), adenosine triphosphate (ATP) content, and viability as determined by formazan production and trypan-blue exclusion (>95%). Glucose stimulation index of freshly isolated islets (2.5 +/- 0.4) was significantly decreased after TLM storage (1.8 +/- 0.2, P < .05) but not after OLM storage (2.3 +/- 0.6). Islet transplantation in diabetic nude mice demonstrated sustained graft function in all experimental groups. CONCLUSIONS: This study demonstrates that viable pig islets can be successfully isolated after prolonged ischemia utilizing PFC alone for oxygenation of cold-stored pig pancreases. The easy handling of OLM could facilitate the regular application of PFC as pancreas preservation solution.

Adenosine↗
PubMed1996

Matrix alters the proliferative response of enterocytes to growth factors.

BACKGROUND: Enterocyte growth and differentiation along the crypt-villus axis is a highly controlled process. Crypt cells divide to produce enterocytes that migrate along the villus axis prior to terminal differentiation. Growth factors and basement membrane may be involved in the regulation of this process. METHODS: IEC-6 proliferation was measured in cells grown on laminin or collagen I by measuring the conversion of Owen's reagent to formazan. Cells were grown with no growth factor, one growth factor (ie, epidermal growth factor, insulin-like growth factor-I, or transforming growth factor-alpha), or a combination of factors. RESULTS: Enterocyte proliferation was increased in all groups given mitogenic growth factors with additive increases in those given combinations. Enterocytes grown on laminin showed a significantly decreased proliferative response to mitogenic growth factors in all experimental groups. Matrix did not alter seeding efficiency or cell viability. CONCLUSION: Basement membrane may play a role in the regulation of cell proliferation along the crypt-villus axis.

Animals↗
PubMed2002

Is reduction of the sulfonated tetrazolium 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2-tetrazolium 5-carboxanilide a reliable measure of intracellular superoxide production?

The sulfonated tetrazolium 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2-tetrazolium 5-carboxanilide (XTT) is advantageous in that it yields a water-soluble formazan, unlike most other available tetrazoliums. XTT is reducible by superoxide, as are other tetrazoliums, but is not directly reduced by xanthine oxidase plus xanthine or by glucose oxidase plus glucose. This led to the suggestion that XTT reduction might serve as a reliable index of intracellular O(2)(-) production. We now show that soluble extracts of Escherichia coli contain two NADPH:XTT reductases that act aerobically or anaerobically. That being the case, XTT reduction is not a reliable measure of intracellular O(2)(-).

Catalase↗
PubMed2003

A colorimetric assay for inorganic pyrophosphate that is also useful for measuring product accumulation in polymerase chain reactions.

A novel coupled enzyme assay for measuring inorganic pyrophosphate (PP(i)) in biological samples is described. The total PP(i) is determined by a reaction with inosine 5'-monophosphate, catalyzed by hypoxanthine-guanine phosphoribosyl transferase, yielding hypoxanthine and phosphoribosyl pyrophosphate. The hypoxanthine is oxidized to uric acid by xanthine oxidase/xanthine dehydrogenase and can be measured by formation of formazan when a tetrazolium salt is used as the oxidant. The method is also useful for detecting and quantifying PP(i) released from nucleotides during polymerase chain reactions. This rapid and simple method for detecting amplified nucleic acids permits low-cost monitoring by eye or spectrophotometer.

Colorimetry↗
PubMed2002

Carbazolequinone induction of caspase-dependent cell death in Src-overexpressing cells.

We previously reported that RSV-transformed quail neuroretina cells (QNR-ts68) were highly resistant to apoptosis provoked by serum withdrawal, and that this property was due to v-Src kinase activity. The present study investigates the cytotoxic effect and the functional mechanism of carbazolequinone-mediated cell death in this system. QNR-ts68 cells were subjected to carbazolequinone treatment and both growth inhibition and cell death induction were examined using formazan assays. Cell death mechanism (both apoptosis and necrosis) was confirmed through phosphatidyl serine exposure and propidium iodide incorporation. Furthermore, the effect of active carbazolequinone was inhibited by a pan caspase inhibitor. Cytofluorimetric and immunofluorescence data demonstrated the activation of caspase-3 and the involvement of mitochondria. Therefore, this study clearly indicates that carbazolequinones could induce cell death in transformed cells displaying high levels of antiapoptotic tyrosine kinase activity. Further investigations would be necessary to elucidate the mechanisms by which these carbazolequinones act as antitumor agents.

Amino Acid Chloromethyl Ketones↗
PubMed1997

Evidence for differential roles of the Rho subfamily of GTP-binding proteins in glucose- and calcium-induced insulin secretion from pancreatic beta cells.

We utilized clostridial toxins (with known specificities for inhibition of GTPases) to ascertain the contribution of candidate GTPases in physiologic insulin secretion from beta cells. Exposure of normal rat islets or isolated beta (HIT-T15) cells to Clostridium difficile toxins A and B catalyzed the glucosylation (and thereby the inactivation) of Rac, Cdc42, and Rho endogenous to beta cells; concomitantly, either toxin reduced glucose- or potassium-induced insulin secretion from rat islets and HIT cells. Treatment of beta cells with Clostridium sordellii lethal toxin (LT; which modified only Ras, Rap, and Rac) also reduced glucose- or potassium-induced secretion. However, clostridial toxin C3-exoenzyme (which ADP-ribosylates and inactivates only Rho) was without any effect on either glucose- or potassium-induced insulin secretion. These data suggest that Cdc42, Rac, Ras, and/or Rap (but not Rho) may be needed for glucose- or potassium-mediated secretion. The effects of these toxins appear to be specific on stimulus-secretion coupling, since no difference in metabolic viability (assessed colorimetrically by quantitating the conversion of the tetrazolium salt into a formazan in a reduction reaction driven by nutrient metabolism) was demonstrable between control and toxin (A or LT)-treated beta cells. Toxin (A or LT) treatment also did not alter glucose- or potassium-mediated rises in cytosolic free calcium concentrations ([Ca2+]i), suggesting that these GTPases are involved in steps distal to elevations in [Ca2+]i. Recent findings indicate that the carboxyl methylation of Cdc42 is stimulated by only glucose, whereas that of Rap (Kowluru et al., J Clin Invest 98: 540-555, 1996) and Rac (present study) are regulated by glucose or potassium. Together, these findings provide direct evidence, for the first time, that the Rho subfamily of GTPases plays a key regulatory role(s) in insulin secretion, and they suggest that Cdc42 may be required for early steps in glucose stimulation of insulin release, whereas Rap and/or Rac may be required for a later step(s) in the stimulus-secretion coupling cascade (i.e. Ca2+-induced exocytosis of insulin).

ADP Ribose Transferases↗
PubMed1978

Reactivity of antiinflammatory and superoxide dismutase active Cu(II)-salicylates.

The activity of chelated Cu(II) with four different aspirin-like drugs in various superoxide dismutase assays was examined. Prior to these studies the oxidation state of the involved copper was measured by x-ray photoelectron spectrometry and was found to be +II throughout. All copper complexes were able to suppress the xanthine-xanthine oxidase mediated reduction of both cytochrome c and nitroblue tetrazolium as well as the formazan formation by KO2 in a specific manner. The hydroxylation of benzo-[alpha]-pyrene as well as the demethylation of 7-ethoxycoumarin using induced hepatic rat microsomes could be successfully inhibited by the employed Cu(II) chelates. Cu(II)-acetylsalicylate was the most active copper complex. Our findings support the proposal that Cu(II) chelates are the active forms of aspirin-like antiinflammatory agents.

Animals↗
PubMed1986

An automated colorimetric microassay for neuronotrophic factors.

A microassay is described for determining the number of neurons surviving after 24 h in response to added neuronotrophic factors. Neuronal cultures in 96-well microtiter plates are supplied with a yellow tetrazolium derivative, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), which is taken up selectively by viable neurons and converted to a blue formazan product. The amount of blue color development can be rapidly quantified using an automatic microplate spectrophotometer. The resulting optical density is directly proportional to the number of viable neurons. The spectrophotometer has been interfaced with a computer allowing a print out of individual absorbance values and calculation of half-maximal (one trophic unit) neuronal survival. The assay has been used for the quantification of the trophic activities of nerve growth factor and ciliary neuronotrophic factor using, respectively, dorsal root and ciliary ganglionic neurons from 8-day chick embryos. Assay parameters were optimized so that about 2000 individual cultures of ganglionic neurons can be set up and analyzed each day, thus allowing the serial titration in duplicate of 80-120 separate samples. The determination of neuronal number and titer calculation steps now requires about 2 min per microplate (96 cultures), a 50-fold reduction in time over existing methods.

Animals↗
PubMed1996

Pituitary adenylate cyclase-activating polypeptide protects rat-cultured cortical neurons from glutamate-induced cytotoxicity.

We have investigated the effects of pituitary adenylate cyclase-activating polypeptide with 38 residues (PACAP38) on glutamate-induced neuronal cell death in rat-cultured cortical neurons. The rat-cultured neurons were obtained from E17 day-old embryos and cultured in a chemically defined medium without serum for 10 days, after which more than 95% of the cells were stained by a specific antibody against MAP-2, a specific marker for neurons. The number of viable neurons was identified by the mitochondrial conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to formazan, which was detected by the associated change in optical density at 570 nm. Glutamate-induced neuronal cell death was suppressed by PACAP38 at concentrations as low as 10(-13) M, and at 10(-11) M maximally suppressed half of the amount of glutamate-induced cell death seen in a control situation (no PACAP38). The dose-response curve was bell-shaped. Dibutyryl cAMP (dbcAMP) also increased the number of neurons that were protected from damage with a bell-shaped dose-response curve suggesting that PACAP exerts its neuroprotective effect through the activation of a cAMP signal transduction system. However, cAMP accumulation in the media of neurons was stimulated by PACAP38 at concentrations as low as 10(-11) M, a much higher concentration than the minimal effective dose of PACAP38 required for protection against glutamate-induced neuronal cell death. Among the three neuropeptides of PACAP38, arginine vasopressin (AVP) and C-type natriuretic peptide (CNP), only PACAP38 exhibited a neurotrophic effect in the glutamate-induced neuronal cell death at the indicated concentrations. These data indicate that PACAP38 is one of the more important neuroprotective factors. The kind of intracellular signal transduction system involved in the neuroprotective effect of PACAP38 still remains to be established.

Animals↗
PubMed1996

Localization of nitric oxide synthase in the goldfish retina.

The localization of nitric oxide synthase and NADPH-diaphorase was studied in the goldfish retina by means of immunohistochemistry or tetrazolium salt technique. Nitric oxide synthase was found in some small neurons of the inner nuclear layer and in large neurons of the ganglion cell layer. The reaction product was localized in the outer plexiform layer and a diffuse labeling was also observed in the inner plexiform layer. In addition to the outer segments of photoreceptors, NADPH-diaphorase labeled several neurons of the inner nuclear layer and some neurons scattered in the ganglion cell layer. Both outer and inner plexiform layers were labeled. Ultrastructural observations showed that the reaction product was found to be bound to the endoplasmic membranes of positive neurons. In the outer plexiform layer the formazan precipitate labeled prevailingly the presynaptic terminals of rods and cones, in the inner plexiform layer both pre- and postsynaptic profiles showed the reaction product.

Animals↗
PubMed1999

High glucose-6-phosphate dehydrogenase activity contributes to the structural plasticity of periglomerular cells in the olfactory bulb of adult rats.

Glucose-6-phosphate dehydrogenase (G6PD) activity, assayed spectrophotometrically, was found to be higher in the olfactory bulb (OB) than in other brain areas of adult rats [P. Ninfali, G. Aluigi, W. Balduini, A. Pompella, Glucose-6-phosphate dehydrogenase is higher in the olfactory bulb than into other brain areas, Brain Res. 744 (1997) 138-142]. Histochemical demonstration of G6PD activity in cryostat sections of OB, analyzed with optical microscopy, revealed a marked and well defined line of formazan deposition in the internal part of the glomerular layer (Glm), indicating that G6PD was much higher in cells distributed along the glomeruli. Electron microscope analysis showed that G6PD activity was mainly concentrated in cytoplasm and dendrites of periglomerular cells, the interneurons which span glomeruli and connect olfactory nerves with mitral/tufted cells. Since G6PD regulates the flux through the hexose monophosphate shunt (HMS) pathway, which provides NADPH for reductive biosynthesis and pentose phosphates for nucleic acid formation, it can be concluded that high G6PD activity in periglomerular neurons is functional to their differentiating capability. This result is consistent with the occurrence of structural plasticity events in the OB of adult rats.

Animals↗
PubMed2000

Preconditioning the human myocardium by simulated ischemia: studies on the early and delayed protection.

BACKGROUND: There are data supporting the existence of ischemic preconditioning in man. This study investigated the most effective preconditioning protocol for the human myocardium and whether the second window of ischemic preconditioning (24 h) is as protective as the first window (< or = 2 h). METHODS AND RESULTS: Right atrial appendages (n = 6/group) obtained during coronary bypass surgery were prepared and superfused with normoxic and normothermic Krebs-Henseleit solution. After 30 min stabilisation, muscles were subjected to various preconditioning protocols followed by 90 min ischemia and 120 min reperfusion. At the end of each protocol, the leakage of creatinine kinase (CK, U/g wet wt) and the reduction of MTT to insoluble formazan dye (OD/mg wet wt), an index of cell viability, were measured. In study 1, preconditioning was induced by 2, 3, 5 and 10 min of ischemia followed by 5 min reperfusion. In study 2, 1-4 cycles of 2 or 5 min ischemia-5 min reperfusion were applied. In study 3, preconditioning was induced by 5 min ischemia-5 min reperfusion followed by 1, 2, 3 or 4 h reperfusion before the subsequent 90 min ischemia. In study 4, preconditioning with 5 min ischemia followed by 5 min reperfusion either immediately preceded 30 or 90 min ischemia/120 min reperfusion or was applied 24 h before. In study 1 and 2, optimal protection was achieved with 5 min or two cycles of 2 min preconditioning ischemia (CK = 3.06 +/- 0.31 and 2.89 +/- 0.02; MTT = 0.56 +/- 0.05 and 0.47 +/- 0.09, respectively vs. CK = 5.56 +/- 0.52 and MTT = 0.18 +/- 0.04 in ischemia alone group; P < 0.05). In study 3, protection was observed 2 h after preconditioning (CK = 3.43 +/- 0.22 and MTT = 0.46 +/- 0.09; P < 0.01 vs. ischemia alone group) but it was lost beyond 2 h (CK = 6.30 +/- 0.56 and MTT = 0.16 +/- 0.02 after 3 h; P = NS vs. ischemia alone group). In study 4, protection was observed 24 h following preconditioning when the atrial specimens were exposed to 30 min ischemia (CK = 2.96 +/- 0.38 and MTT = 0.61 +/- 0.01 vs. CK = 4.56 +/- 0.26 and MTT = 0.43 +/- 0.02 in ischemia alone group, P < 0.05); however, when the period of ischemia was extended to 90 min the beneficial effect of preconditioning was lost (CK = 10.28 +/- 0.05 and MTT = 0.11 +/- 0.05 vs. CK = 9.56 +/- 0.62 and MTT = 0.104 +/- 0.05 in ischemia alone group, P = NS). CONCLUSIONS: In the isolated human myocardium maximal protection induced by preconditioning is achieved by a total 4-5 min ischemic stimulus, an effect that is lost beyond 2 h of its application. Two windows of protection were identified, the first (< or = 2 h) being more potent than the second (24 h).

Analysis of Variance↗
PubMed2001

Effects of 3-methylcholanthrene and aspirin co-administration on ALDH3A1 in HepG2 cells.

The effects of two different protocols of 3-methylcholanthrene (3MC) and aspirin co-administration were studied in a well-established human hepatoma cell line (HepG2). During this work, we have performed toxicity tests for cell viability/cell proliferation as well as studies on the expression of ALDH3A1 after exposure of HepG2 cells to 3MC or/and aspirin. For the evaluation of toxic concentrations of 3MC and aspirin, the WST-1 test was used. WST-1 is a reliable cytotoxicity test which is based on the cleavage of the tetrazolium salt WST-1 to formazan by mitochondrial enzymes of living cells. A broad range of drug concentrations for either 3MC (0.25-50.0 microM) or aspirin (0.05-10.0 mM) were used for cell exposure, in several periods of time. The expression of ALDH3A1 in HepG2 cells showed typical time- and dose-response curves of induction after application of 3MC (1-5 days, 1.5-5.0 microM, respectively). When cells were firstly exposed to 3MC (2.5 and 5.0 microM) and then to aspirin (0.25 mM), the induced ALDH3A1 activity was further enhanced in a statistically significant way (P<0.05). On the contrary, when aspirin application was preceded 3MC exposuring a statistically significant decrease in ALDH3A1 inducibility was observed, as compared with the application of 3MC alone.

Aldehyde Dehydrogenase↗
PubMed2002

Determination of superoxide dismutase and glutathione peroxidase activities in blood of healthy pediatric subjects.

BACKGROUND: Given the growing requirement of antioxidant enzymes measurements in laboratory and the increasing role of SOD/GPx ratio in the balance of reactive oxygen species (ROS), the aim of our study was to contribute to define reference values of enzyme activities in Italian healthy children, by determining SOD in erythrocytes and GPx in whole blood. METHODS: SOD (E.C.1.15.1.1) and GPx (E.C.1.11.1.9) activities were spectrophotometrically assayed in erythrocytes with commercial kits. SOD activity was expressed as the amount of protein causing a 50% inhibition of formazan dye (505 nm), employing xanthine and xanthine oxidase to generate superoxide radicals. Units of GPx activity were calculated following NADPH oxidation at 340 nm using cumene hydroperoxide as the substrate. RESULTS: Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were assayed in blood of 45 healthy pediatric subjects (26 males and 19 females between 0 and 14 years of age). We found no significant differences in blood antioxidant enzymes both in all population and when we distributed the subjects for age classes and sex. CONCLUSION: the definition of pediatric values of antioxidant enzyme activities in blood of healthy children may be useful in monitoring SOD and GPx in physiologic and pathologic conditions, also for future therapeutic trials.

Adolescent↗
PubMed2000

A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1).

A simple and reproducible microtiter plate assay for measuring superoxide dismutase (SOD) activity is described. Water-soluble tetrazolium, the sodium salt of 4-[3-(4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-be nzene disulfonate, was used as a detector of superoxide radical generated by xanthine oxidase and hypoxanthine, in the presence of a range of concentrations of superoxide dismutase. A major advantage of the assay is that one reaction mixture is prepared and aliquotted into wells, avoiding pipetting errors and variable xanthine oxidase activity between samples. Inclusion of standardized SOD solution in each run enables inter-assay comparability without requiring a constant superoxide generation rate under all occasions. The assay is applicable for chloroform-ethanol red cell extracts as well as tissue homogenates without high-speed centrifugation. Fifty percent inhibition of formazan formation was achieved at 2.4+/-0.1 ng of Cu, ZnSOD per well with the coefficient of variation 4.2%.

Animals↗