Formazan Production Research Articles

Enhanced tetrazolium violet reduction of Salmonella spp. by magnesium addition to the culture media

Tetrazolium salts (TTZ), such as tetrazolium violet (TV), have been widely used for microbiological studies. The formation of the colored formazan product due to bacterial reduction of the uncolored reagent is extensively exploited to stain cells or colonies in agar or on filters. But an important toxic effect of tetrazolium salts on bacteria exists that limits their use at high concentrations, impairing the efficient staining of the colonies. This is especially the case for Salmonella spp. where we observed, using a classic photometric approach and mathematical modeling of the growth, an important impact of tetrazolium violet on the apparent growth rate below the inhibitory concentration. In this study, we demonstrate that adding magnesium to the medium in the presence of TV leads to a significant increase in the apparent growth rate. Moreover, when higher TV concentrations are used which lead to total inhibition of Salmonella strains, magnesium addition to the culture media allows growth and TV reduction. This effect of magnesium may allow the use of higher TTZ concentrations in liquid growth media and enhance bacteria detection capabilities.

Antiviral efficacy of nine nucleoside reverse transcriptase inhibitors against feline immunodeficiency virus in feline peripheral blood mononuclear cells

To compare cytotoxic effects and antiviral efficacy of 9 nucleoside reverse transcriptase inhibitors (NRTIs) against FIV in feline peripheral blood mononuclear cells. Peripheral blood mononuclear cells obtained from 3 specific pathogen-free cats. 3 of the 9 NRTIs had not been previously assessed in feline cell lines. Cytotoxic effects were determined by colorimetric quantification of a formazan product resulting from bioreduction of a tetrazolium reagent by viable peripheral blood mononuclear cells; uninfected cells from 1 cat were used in these assays. Cells from all 3 cats were infected with a pathogenic clone of FIV, and in vitro antiviral efficacy of each NRTI was assessed with an FIV p24 antigen capture ELISA. Cytotoxic effects in feline peripheral blood mononuclear cells were observed only at concentrations > 10 μM for all 9 NRTIs. Comparison of the cytotoxic effect at the highest concentration investigated (500 μM) revealed that didanosine and amdoxovir were significantly less toxic than abacavir. All drugs induced a dose-dependent reduction of FIV replication. At the highest concentration investigated (10 μM), there was no significant difference in antiviral efficacy among the test compounds. The evaluated NRTIs had low cytotoxicity against feline peripheral blood mononuclear cells and appeared to be safe options for further in vivo evaluation for the treatment of FIV-infected cats. There was no evidence suggesting that the newly evaluated compounds would be superior to the existing NRTIs for reducing FIV burden of infected cats.

Cytotoxicity of halogenated graphenes

Graphene and its family of derivatives possess unique and remarkable physicochemical properties which make them valuable materials for applications in many areas like electronics, energy storage and biomedicine. In response to the possibility of its large-scale manufacturing as commercial products in the future, an investigation was conducted to determine the cytotoxicity of one particular family of graphene derivatives, the halogenated graphenes, for the first time. Halogenated graphenes were prepared through thermal exfoliation of graphite oxide in gaseous chlorine, bromine or iodine atmospheres to yield chlorine- (TRGO-Cl), bromine- (TRGO-Br) and iodine-doped graphene (TRGO-I) respectively. 24 h exposure of human lung carcinoma epithelial cells (A549) to the three halogenated graphenes and subsequent cell viability assessments using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and water-soluble tetrazolium salt (WST-8) assays revealed that all the halogenated graphenes examined are rather cytotoxic at the concentrations tested (3.125 μg mL(-1) to 200 μg mL(-1)) and the effects are dose-dependent, with TRGO-Cl reducing the cell viability to as low as 25.7% at the maximum concentration of 200 μg mL(-1). Their levels of cytotoxicity can be arranged in the order of TRGO-Cl > TRGO-Br > TRGO-I, and it is suggested that the amount of halogen present in the graphene material is the determining factor for the observed trend. Control experiments were carried out to test for possible nanomaterial-induced interference as a consequence of reaction between the halogenated graphenes and the viability markers (MTT/WST-8 reagent) or binding of the formazan products under cell-free conditions. The data obtained eliminate the probability of significant influence by these interferents as the change in the normalized percentage of formazan formed is relatively small and thorough washings were performed prior to the viability assessments to reduce the amount of halogenated graphenes that could eventually interact with the MTT/WST-8 assays. More studies need to be carried out in the future to complement the results obtained in this initial study in an attempt to develop a better understanding of the health hazards that the halogenated graphenes pose.

Protective effect of aronia melanocarpa fruit juice in a model of cisplatin-induced cytotoxicity in vitro

The aim of the present study was to investigate the protective potential of Aronia melanocarpa fruit juice in a model of cisplatin-induced cytotoxicity in the human embryonal kidney cell line HEK293T. The cellular viability was assessed using the MTT-dye reduction assay based on the reduction of the yellow tetrazolium dye MTT to a violet formazan product via the mitochondrial succinate dehydrogenase in viable cells. Cisplatin was applied in various concentrations either alone or after a 24-hour pretreatment of the cells with Aronia melanocarpa fruit juice at 0.1 and 0.05 mg/ml. The half maximal inhibitory concentrations (IC50 values) were derived from the concentration-response curves to cisplatin. Applied alone, the anticancer drug caused a prominent decrease of cellular viability with IC50 8.3 +/- 1.1 microM. The juice proved to significantly ameliorate the in vitro cytotoxicity of the platinum drug, in a concentration-dependent manner. The pretreatment of the cells with Aronia melanocarpa fruit juice resulted in a significant increase (p < 0.001) of IC50 for cisplatin to 25.1 +/- 2.7 microM (at 0.05 mg/ml) and 34.4 +/- 3.4 microM (at 0.1 mg/ml), respectively. The protective effect of Aronia melanocarpa fruit juice observed in this study is most probably due to its well appreciated antioxidant activity as oxidative stress plays a central role in the toxic effects of cisplatin.

ΔNp63 expression in four carcinoma cell lines and the effect on radioresistance—a siRNA knockdown model

This study investigated the expression of ΔNp63α in carcinoma cell lines of the upper aerodigestive tract and their potential influence on radioresistance using a small interfering RNA (siRNA) knockdown approach. Four carcinoma cell lines were investigated for the expression of the ΔNp63 isoform by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (0, 24, 48 h) with and without single dose irradiation of 6 Gy. Furthermore, all cell lines were transfected with siRNA against the ΔNp63α isoform over 24 h. Knockdown effectiveness was controlled by qRT-PCR and Western blot. Apoptotic events were evaluated by terminal transferase dUTP nick end labeling (TUNEL) assay and cross-checked by a test for cell viability (WST-1, Roche) over 48 h. All cell lines presented varying expression of the ΔNp63α isoform with and without irradiation. A sufficient knockdown rate was established by siRNA transfection. Knockdown of the ΔNp63 isoform showed an effect on radiation sensitivity proven by an increase of apoptotic events detectable by immunofluorescence (TUNEL assay) and likewise a significant reduction of formazan production (WST-1 test) in three cell lines. We found overexpression of ΔNp63α with and without irradiation in three cell lines, and the knockdown of ΔNp63α led to increased apoptotic events and fewer viable cells. Thus, the overexpression of ΔNp63α might protect carcinoma cells against irradiation effects. The present work supports the hypothesis that protein 63 might serve as a negative predictor for irradiation response and survival in a clinical setting and may be a target for future therapeutic strategies.

The noncellular reduction of MTT tetrazolium salt by TiO2 nanoparticles and its implications for cytotoxicity assays

We report results of noncellular tests, revealing the occurrence of photocatalytic interactions between titanium dioxide (TiO2, titania) nanoparticles and the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide] cytotoxicity indicator. These interactions induce the reduction of MTT and formation of purple formazan under biologically relevant conditions. Classical MTT assays have been performed to evaluate the production of formazan in DMEM-F12 and RPMI-1640 cell culture media (containing 10% fetal bovine serum-FBS) treated with Degussa-P25 TiO2 nanoparticles, in the absence of cells. The colorimetric determinations revealed the noncellular MTT to formazan transformation induced by TiO2 nanoparticles, under conditions commonly used for in vitro cytotoxicity testing of nanomaterials. The formazan precipitation was found to be proportional to the TiO2 concentration, being enhanced under laboratory daylight exposure. The photocatalytic nature of the studied effect was assessed under UV irradiation at 365nm. The biological significance of the reported reaction was established with respect to cellular reference experiments performed on V79-4, HeLa and B16 cell lines. The results show false viability increases with up to 14% (for TiO2 concentrations generally higher than 50μg/ml), induced by the TiO2–MTT reaction. This type of artifacts may lead to underestimated toxicity or false proliferation results.

The Inhibitory Role of Hydrogen Sulfide in Airway Hyperresponsiveness and Inflammation in a Mouse Model of Asthma

Cystathionine γ-lyase (CSE) is one of the major enzymes producing hydrogen sulfide (H2S) in lungs, participating in the regulation of respiratory functions. The role of CSE-derived H2S in eosinophil-dominant inflammation in allergic diseases has been unclear. The objective of this study was to explore the protective role of H2S against allergen-induced airway hyperresponsiveness (AHR) and inflammation. CSE expression and H2S production rate were assessed in mouse lung tissues with ovalbumin (OVA)-induced acute asthma. AHR, airway inflammation, and Th2 response in wild-type (WT) mice were compared with those in CSE gene knockout (KO) mice. The effect of NaHS, an exogenous H2S donor, was also evaluated on these parameters. CSE expression was absent and H2S production rate was significantly lower in the lungs of CSE KO mice when compared with WT littermates. OVA challenge decreased lung CSE expression and H2S production in WT mice. CSE deficiency resulted in aggravated AHR, increased airway inflammation, and elevated levels of Th2 cytokines such as IL-5, IL-13, and eotaxin-1 in bronchoalveolar lavage fluid after OVA challenge. The aforementioned alterations were reversed by exogenous H2S treatment. More importantly, NaHS supplement rescued CSE KO mice from the aggravated pathological process of asthma. The CSE/H2S system plays a critical protective role in the development of asthma. A new therapeutic potential for asthma via targeting CSE/H2S metabolism is indicated.

Immunotoxic and genotoxic potential of arsenic and its chemical species in goats

The study investigated the immunotoxic and genotoxic effect of arsenic and its different species on goats. It was found that arsenic causes haematological crisis. Histopathological changes in spleen and reduced serum immunoglobulin G level without any changes in formazan production in arsenic-treated animals indicated that arsenic is toxic to the humoral immune system. Increased caspase-3 production and higher number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)-positive bone marrow cells along with oligonucleosomal DNA fragmentation on agarose gel suggested apoptosis induction by arsenic in the bone marrow cells of goat. Total arsenic concentration in the plasma, bone marrow, and spleen of the exposed group was, respectively, 1.22 ± 0.11, 2.20 ± 0.21, and 3.39 ± 0.14 ppm. Speciation study revealed that arsenite and organoarsenic were the major arsenic species in these samples, suggesting their role in immunotoxic and genotoxic potential in goats.

Flavocytochromeb2-Based Enzymatic Method of L-Lactate Assay in Food Products

L-lactate, a key metabolite of the anaerobic glycolytic pathway, plays an important role as a biomarker in medicine, in the nutritional sector and food quality control. For these reasons, there is a need for very specific, sensitive, and simple analytical methods for the accurate L-lactate measuring. A new highly selective enzymatic method for L-lactate determination based on the use of flavocytochrome b 2 (EC 1.1.2.3; FC b 2) isolated from the recombinant strain of the yeast Hansenula polymorpha has been developed. A proposed enzymatic method exploits an enzymatic oxidation of L-lactate to pyruvate coupled with nitrotetrazolium blue (NTZB) reduction to a colored product, formazan. The maximal absorption peak of the colored product is near λ = 525 nm and the linear range is observed in the interval 0.005–0.14 mM of L-lactate. The main advantages of the proposed method when compared to the LDH-based routine approaches are a higher sensitivity (2.0 μM of L-lactate), simple procedure of analysis, usage of inexpensive, nontoxic reagents, and small amount of the enzyme. Enzymatic oxidation of L-lactate catalyzed by flavocytochrome b 2 and coupled with formazan production from nitrotetrazolium blue was shown to be used for L-lactate assay in food samples. A high correlation between results of the proposed method and reference ones proves the possibility to use flavocytochrome b 2-catalysed reaction for enzymatic measurement of L-lactate in biotechnology and food chemistry.

Kinetics of reactive oxygen species produced by rainbow trout (Oncorhynchus mykiss) leukocytes and the effect of the antioxidant astaxanthin

Reactive oxygen species contribute to kill of microorganisms. Their activity is usually measured by their capacity to reduce nitroblue tetrazolium into formazan. The incubation time to allow nitroblue tetrazolium reduction by reactive oxygen species usually ranges from 30 to 60 min. The aim of our study was to determine the kinetics of formazan formation, to determine the shortest incubation time possible, and to find if astaxanthin negatively affects the availability of reactive oxygen species (and defense mechanisms of fish). The blood/nitroblue tetrazolium method is based on nitroblue tetrazolium reduction into formazan by reactive oxygen species present in blood. Formazan can be spectrophotometrically measured, allowing quantification of reactive oxygen species. Reactive oxygen species were measured in blood samples from 30 trout intramuscularly injected with astaxanthin (0.3 mg/100 g of fish) (experimental group) and 30 non-injected trout (controls). Results indicated that in trout non-treated with astaxanthin, the plateau of formazan production was reached after 20 min of incubation. Trout intramuscularly injected with astaxanthin showed the following: on Day 1 after astaxanthin injection, the kinetics were slower but finally reached a plateau similar to astaxanthin-free trouts, and by Day 11 the plateau was significantly higher after 60 min incubation. In conclusion, the kinetics curves here reported allow reducing incubation time of the method to only 20 min in antioxidant-free trout and, on the other hand, our results also revealed that astaxanthin can be used to improve flesh colour in salmonids without affecting reactive oxygen species availability and therefore the defense mechanisms of trout.

Overexpression of Human Arginine Decarboxylase Rescues Human Mesenchymal Stem Cells against H2O2Toxicity through Cell Survival Protein Activation

In this study, we explored the potentiality of human arginine decarboxylase (ADC) to enhance the survival of mesenchymal stem cells (MSCs) against unfavorable milieu of host tissues as the low survival of MSCs is the issue in cell transplantation therapy. To address this, human MSCs overexpressing human ADC were treated with H2O2 and the resultant intracellular events were examined. First, we examined whether human ADC is overexpressed in human MSCs. Then, we investigated cell survival or death related events. We found that the overexpression of human ADC increases formazan production and reduces caspase 3 activation and the numbers of FITC, hoechst, or propidium iodide positive cells in human MSCs exposed to H2O2. To elucidate the factors underlying these phenomena, AKT, CREB, and BDNF were examined. We found that the overexpression of human ADC phosphorylates AKT and CREB and increases BDNF level in human MSCs exposed to H2O2. The changes of these proteins are possibly relevant to the elevation of agmatine. Collectively, our data demonstrate that the overexpression of human ADC stimulates pro-survival factors to protect human MSCs against H2O2 toxicity. In conclusion, the present findings support that ADC can enhance the survival of MSCs against hostile environment of host tissues.

Seed Germination and Viability Test in Tetrazolium (TZ) Assay

Tetrazolium (TZ) assay is the fast evaluation for seed viability and alternative quick method for seed’s germinability (Porter et al., 1947; Wharton, 1955). All respiring tissues are capable of converting a colourless compound, TZ (2,3,5 triphenyl tetrazolium chloride) to a carmine red coloured water-insoluble formazan by hydrogen transfer reaction catalysed by the cellular dehydrogenases. TZ enters both living and dead cells but only living cells catalyse the formation of formazan which being non-diffusible stains the viable seeds red whereas the absence of respiration prevents formazan production making the dead seeds (aged tissue) remain unstained.The brief description of this protocol has been reported in Verma et al., 2013.

Improved 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay for measuring the viability of lactic acid bacteria

The aim of this study was to adapt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay (MCA) (Wang et al., 2010) for determination of lactic acid bacteria cell count. Our study is helpful in developing protocols for measuring the viability of other lactic acid bacteria. We determined the cell concentration range which gives linear regression of CFU (colony forming units) and formazan production. In our experiment three authentic lactic acid bacteria strains were investigated: Lactobacillus rhamnosus VT1, L. plantarum 2142, and L. sakei DSM 2001. As data show beside the strain also the growth medium has influence on the MTT reduction activity, this means that for each special case separate calibration curve has to be prepared. Based on MTT reduction we also developed an improved microtiter plate assay which proved to be reliable for a rapid cell count determination. So our methods are only applicable for estimate cell number with fixed parameters, but under given circumstances they are fast and sensitive methods. With the modified methods we can rapidly measure the dehydrogenase enzyme activity of the lactic acid bacteria cells. With the microplate assay we can measure many conditions and strains at the same time.

Simplified detection of food-borne pathogens: An in situ high affinity capture and staining concept

Food industries need simple, rapid and cost-effective solutions for pathogen detection in food and environmental samples. In this paper, we describe a simple but novel detection concept combining an affinity capture surface and intracellular metabolic marker to visualize the bacterial presence on the affinity surface. The surface of a Solid Phase Support (SPS) is functionalized with specific phage tail proteins targeted to the bacterial pathogen of interest. The SPS is placed directly into the primary food enrichment bag after stomaching. Following incubation, the captured bacteria are visually detected in situ as a result of the bacterial reduction of the colorless soluble substrate triphenyltetrazolium chloride (TTC) (present in the primary culture medium) to an intracellular red insoluble formazan product. Detection on the SPS is observed as an intense red color after 22 to 40hours of enrichment. This is not impaired by the presence of food particles and the natural background microflora. The in situ method significantly simplifies pathogen detection by eliminating any post-enrichment intervention that is necessary in the traditional methods of analysis. We have demonstrated the application of this new approach for the detection of Escherichia coli O157: H7, Listeria spp. and Salmonella spp. in artificially contaminated food samples.

Effects of chronic cocaine in rat C6 astroglial cells

Investigations with astroglial cells carry equal importance as those with neurons in drug abuse studies. The present study was aimed to investigate the effect of chronic cocaine administration on cell viability, nitric oxide (NO) production, general respiratory status of mitochondria and total protein levels in rat astroglioma cells after 24 h of treatment. In addition, the effect of cocaine was assessed for 24 h on brine shrimp larvae in order to study their sensitivity to the drug. It was observed that cocaine caused a significant dose-dependent decrease in astroglial cell viability with an LC50 of 4.717 mM. It was found that cocaine did not induce or inhibit NO production in the cells. Evaluation of mitochondrial dehydrogenase activity in terms of formazan production in astroglial cells indicated that cocaine significantly interfered with the general respiratory status of mitochondria with an ED50 of 6.153 mM. Furthermore, cocaine was shown to deplete the total protein levels in the cells with an ED50 of 5.435 mM. In vivo study with brine shrimp larvae showed that these larvae were highly sensitive to cocaine with an ED50 of 2.41 mM. In summary, our findings suggest that cocaine-induced cytotoxicity in the cells was non-specific. The cumulative effect arising from the significant loss of respiration and total cellular proteins is the cause of astroglial cell death.

XTT assay of ex vivo saliva biofilms to test antimicrobial influences.

Objective: Many dental diseases are attributable to biofilms. The screening of antimicrobial substances, in particular, requires a high sample throughput and a realistic model, the evaluation must be as quick and as simple as possible. For this purpose, a colorimetric assay of the tetrazolium salt XTT (sodium 3'-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate) converted by saliva biofilms is recommended. Cleavage of XTT by dehydrogenase enzymes of metabolically active cells in biofilms yields a highly colored formazan product which is measured photometrically.Materials and method: The suitability of the XTT assay for detecting the vitality of ex vivo saliva biofilms was tested to determine the efficacy of chlorhexidine and ozone versus saliva biofilms grown on titanium discs.Results: The XTT method lends itself to testing the vitality of microorganisms in saliva biofilms. The sensitivity of the arrays requires a specific minimum number of pathogens, this number being different for planktonic bacteria and those occurring in biofilms. The antibacterial effect after treatment with chlorhexidine or ozone was measured by XTT conversion that was significantly reduced. The antimicrobial efficacy of 60 s 0.5% and 0.1% chlorhexidine treatment was equal and comparable with 60 s ozone treatment. Conclusion: The XTT assay is a suitable method to determine the vitality in saliva biofilms, permitting assessment of the efficacy of antimicrobial substances. Its quick and easy applicability renders it especially suitable for screening.

MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets

Although MTT is widely used to assess cytotoxicity and cell viability, the precise localization of its reduced formazan product is still unclear. In the present study the localization of MTT formazan was studied by direct microscopic observation of living HeLa cells and by colocalization analysis with organelle-selective fluorescent probes. MTT formazan granules did not colocalize with mitochondria as revealed by rhodamine 123 labeling or autofluorescence. Likewise, no colocalization was observed between MTT formazan granules and lysosomes labeled by neutral red. Taking into account the lipophilic character and lipid solubility of MTT formazan, an evaluation of the MTT reaction was performed after treatment of cells with sunflower oil emulsions to induce a massive occurrence of lipid droplets. Under this condition, lipid droplets revealed a large amount of MTT formazan deposits. Kinetic studies on the viability of MTT-treated cells showed no harmful effects at short times. Quantitative structure–activity relations (QSAR) models were used to predict and explain the localization of both the MTT tetrazolium salt and its formazan product. These predictions were in agreement with experimental observations on the accumulation of MTT formazan product in lipid droplets.

Hydroxyethyl disulfide as an efficient metabolic assay for cell viability in vitro

Cell viability assays have a variety of well known practical and technical limitations. All the available approaches have disadvantages, such as non-linearity, high background and cumbersome protocols. Several commonly used tetrazolium chemicals rely upon generation of a colored formazan product formed by mitochondrial reduction of these compounds via phenazine methosulfate (PMS). However, sensitivity is inherently limited because their reduction relies on mitochondrial bioreduction and cellular transport of PMS, as well as accessibility to tetrazolium chemicals. In this study, we identify hydroxethyldisulfide (HEDS) as an inexpensive probe that can measure cellular metabolic activity without the need of PMS. In tissue culture medium, HEDS accurately quantitated metabolically active live cells in a linear manner superior to tetrazolium based and other assays. Cell toxicity produced by chemotherapeutics (cisplatin, etoposide), oxidants (hydrogen peroxide, acetaminophen), toxins (phenyl arsine oxide, arsenite) or ionizing radiation was rapidly determined by the HEDS assay. We found that HEDS was superior to other commonly used assays for cell viability determinations in its solubility, membrane permeability, and intracellular conversion to a metabolic reporter that is readily transported into the extracellular medium. Our findings establish the use of HEDS in a simple, rapid and low cost assay to accurately quantify viable cells.

Abstract C195: Inhibition of polo-like kinase 1 (PLK1) by BI 2536: Effects on mitotic index, clonogenic capacity, and proliferation in medulloblastoma cell lines.

Abstract Medulloblastoma is the most common central nervous system tumor in childhood. Despite high survival rates (over 50% for most protocols) the treatment for this tumor usually results in severe sequels. Still, in patients younger than 3 years-old radiotherapy cannot be used turning the prognosis even worse. Polo-like kinase 1 (PLK1), a serine-threonine kinase that regulates mitosis progression, has been repeatedly associated with increased cell proliferation and poorer prognosis in several types of cancer. The aim of this study was to evaluate the effects of the inhibition of PLK1 with BI 2536 on mitotic index clonogenic capacity and cell proliferation, on the UW402 and UW473 pediatric medulloblastoma cell lines. Equal numbers of cells were treated with 50, 100 and 150 nM of BI 2536 for 24, 48 and 72 hours. To calculate the mitotic index cells after treatment dropped onto glass slides, allowed to air dry and stained with Giemsa, 1000 cells were counted. To calculated the clonogenic capacity cells were treated, let to grow for 7 days and then the colonies were fixed with methanol and stained with Giemsa. Only colonies with &amp;gt; 50 cells were counted. To analyze the proliferation cells after treatment were let with culture medium containing 10 μL of XTT dye (XTT II; Roche Molecular Biochemicals, Indianapolis, IN), plates were incubated for 2 hours at 37°C and the formazan product was measured at 450 nm by using an iMark microplate reader (Bio-Rad Laboratories). Each experiment was performed in triplicate wells and repeated in three sets of tests. The tests were analyzed by oneway ANOVA followed by Home-Sidak test. Values of p≤0.05 were considered statistically significant. The Inhibition of PLK1 by this drug showed an efficient decrease (p&amp;lt;0.05) on cell proliferation in dose- and time-dependent manner. The clonogenic assay also demonstrated that BI 2536 causes a significant reduction in colony formation at concentrations as low as 5nM (p&amp;lt;0.05). The mitotic index analysis showed a significant accumulation of dividing cells (p&amp;lt;0.05) with a more evident effect after 24h of treatment. Although further studies are needed, our results point to PLK1 as a potential target to improve medulloblastoma treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C195.

An Ion-insensitive cAMP Biosensor for Long Term Quantitative Ratiometric Fluorescence Resonance Energy Transfer (FRET) Measurements under Variable Physiological Conditions

Ratiometric measurements with FRET-based biosensors in living cells using a single fluorescence excitation wavelength are often affected by a significant ion sensitivity and the aggregation behavior of the FRET pair. This is an important problem for quantitative approaches. Here we report on the influence of physiological ion concentration changes on quantitative ratiometric measurements by comparing different FRET pairs for a cAMP-detecting biosensor. We exchanged the enhanced CFP/enhanced YFP FRET pair of an established Epac1-based biosensor by the fluorophores mCerulean/mCitrine. In the case of enhanced CFP/enhanced YFP, we showed that changes in proton, and (to a lesser extent) chloride ion concentrations result in incorrect ratiometric FRET signals, which may exceed the dynamic range of the biosensor. Calcium ions have no direct, but an indirect pH-driven effect by mobilizing protons. These ion dependences were greatly eliminated when mCerulean/mCitrine fluorophores were used. For such advanced FRET pairs the biosensor is less sensitive to changes in ion concentration and allows consistent cAMP concentration measurements under different physiological conditions, as occur in metabolically active cells. In addition, we verified that the described FRET pair exchange increased the dynamic range of the FRET efficiency response. The time window for stable experimental conditions was also prolonged by a faster biosensor expression rate in transfected cells and a greatly reduced tendency to aggregate, which reduces cytotoxicity. These properties were verified in functional tests in single cells co-expressing the biosensor and the 5-HT(1A) receptor.