The MTT Assay: A Method for Error Minimization and Interpretation in Measuring Cytotoxicity and Estimating Cell Viability.

The MTT assay is extensively used, most often to infer a measure of cytotoxicity of treatments to cells. As with any assay though, there are a number of limitations. The method described here is designed with consideration of how the MTT assay fundamentally works to account for, or at least identify, confounding factors in measurements. It also provides a decision-making framework to best interpret and complement the MTT assay to apply it as either a measure of metabolic activity or cell viability.

SIRT1 Protects Against Particulate Matter-Induced Oxidative Stress in Human Corneal and Conjunctival Epithelial Cells.

Purpose: Sirtuin1 (SIRT1) as a hot therapeutic target for oxidative stress-associated diseases that has been extensively studied. This study aimed to determine the changes in SIRT1 expression in particulate matter (PM)-induced corneal and conjunctival epithelial cell damage and explore potential drugs to reduce PM-associated ocular surface injury. Methods: Immortalized human corneal epithelial cells (HCECs) and human conjunctival epithelial cells (HCjECs) were exposed to an ambient PM sample. Cytotoxicity was evaluated by water-soluble tetrazolium salt-8 assay. SIRT1 expression was measured by Western blot analysis. Reactive oxygen species (ROS) production, cell apoptosis, mitochondrial function, and cell senescence were assessed by using 2',7'-dichlorofluorescein diacetate assay, annexin V apoptosis assay, tetramethylrhodamine ethyl ester assay, and senescence ß-galactosidase staining, respectively. Results: PM-induced cytotoxicity of HCECs and HCjECs occurred in a dose-dependent manner. Increased ROS production, as well as decreased SIRT1 expression, were observed in HCECs and HCjECs after 200 µg/mL PM exposure. In addition, PM induced oxidative stress-mediated cellular damage, including cell apoptosis, mitochondrial damage, and cell senescence. Interestingly, SRT1720, a SIRT1 activator, increased SIRT1 expression and decreased ROS production and attenuated PM-induced cell damage in HCECs and HCjECs. Conclusions: This study determined that SIRT1 was involved in PM-induced oxidative stress in HCECs and HCjECs and found that ROS overproduction may a key factor in PM-induced SIRT1 downregulation. The SIRT1 activator, SRT1720, can effectively upregulate SIRT1 expression and inhibit ROS production, thereby reversing PM-induced cell damage. This study provides a new potential target for clinical treatment of PM-associated ocular surface diseases.

High cytotoxicity of betulin towards fish and murine fibroblasts: Is betulin safe for nonneoplastic cells?

BACKGROUND: Betulin, a natural pentacyclic triterpene with the lupane structure that is present in significant amounts in the outer bark of birch, is known for its broad array of biological and pharmacological properties. Betulin has attracted attention as a potential, natural-origin antimicrobial substance. The literature describes it as selectively toxic to neoplastic cells but safe for normal cells. The research aim was to evaluate the basal cytotoxicity of betulin towards fish (BF-2) and murine (NIH/3T3) fibroblasts. We used four colorimetric tests that provide a preliminary evaluation of possible mechanisms of the cytotoxicity of a compound to assess the degree of the toxicity of betulin after 24, 48 and 72 h of incubation with cells: the MTT assay (mitochondrial activity assessment), the NRU assay (lysosomal membrane integrity assessment), the LDH assay (cellular membrane integrity assessment) and the SRB assay (total cellular protein content determination). RESULTS: The results revealed an exceptionally high sensitivity of mitochondria to the effect of betulin, with the other endpoints being less sensitive. Although murine fibroblasts were more vulnerable to the toxic effect of betulin than fish fibroblasts, the betulin CC50 values for both cell lines were comparable with analogous IC50 values determined by other researchers in studies involving cancerous cells. CONCLUSIONS: The results indicate the need to verify the claim about the selective toxicity of betulin towards malignant cells and to conduct safety/toxicity tests before any potential therapeutic use of betulin in veterinary medicine.

A sensitive EZMTT method provides microscale, quantitative and high-throughput evaluation of drug efficacy in the treatment of Mycobacterium tuberculosis infectious diseases.

Drug resistance has become a serious public health problem in mycobacterial infectious diseases. Here, we investigated a water soluble tetrazolium salt (EZMTT)-based detection method to provide an easy, safe and quantitative antimycobacterial susceptibility test (AMST) method, especially for targeting early detection of loss of drug susceptibility in mycobacteria. After a single addition of the EZMTT detection reagent at the inoculation of mycobacteria culture, the AMST was continuously analyzed in a sealed 96-well plate (100 µl), or a sealed tube to ensure biosafety. Using Mycobacterium tuberculosis H37Ra as the model strain, the EZMTT assay was developed with high reproducibility (Z factor of 0.64) for facile measurements of growth and drug susceptibility. In the comparative AMST study, the 7-day EZMTT method identified not only the same set of drug resistance as the other two methods (the 30-day traditional Löwenstein Jensen solid medium assay and the 10-14 day 8 ml Mycobacteria Growth Indicator Tube liquid method), but also additional strains with loss of drug susceptibility. In conclusion, we demonstrated that the EZMTT-based AMST assay in a sealed microtiter plate has great potential for routine use in medical diagnosis and drug screening to battle the unmet medical need in the treatment of multi- and extensive-drug resistant mycobacteria.

Prdx6 is required to protect human corneal epithelial cells against ultraviolet B injury.

BACKGROUND: The protective role of Prdx6 on rat corneal tissue against ultraviolet B injury in vivo has been confirmed previously. We further investigated the function and molecular mechanism of Prdx6 in human corneal epithelial cells under ultraviolet B radiation. METHODS: The experimental groups were designed as follows: (1) Prdx6 RNAi, (2) Prdx6 RNAi + ultraviolet B radiation, (3) normal human corneal epithelial cells, (4) normal human corneal epithelial cells + ultraviolet B radiation, (5) wild-type Prdx6 overexpression, (6) wild-type Prdx6 overexpression + ultraviolet B radiation, (7) mutant-type Prdx6 overexpression, and (8) mutant-type Prdx6 overexpression + ultraviolet B radiation. The cell survival rate was detected by a Thiazolyl Blue Tetrazolium Bromide assay. Apoptosis, reactive oxygen species, and malondialdehyde were detected with a commercial kit. Gene expression was detected by real-time polymerase chain reaction. RESULTS: We found the following results. (1) Compared to normal cells, the survival rates were 32%, 87%, and 58% under ultraviolet B radiation in the Prdx6 interference, wild-type overexpression, and mutant-type overexpression groups, respectively. The survival rates were decreased to 50% at 24 h and 31% at 48 h when the phospholipase A2 activity of Prdx6 was inhibited after ultraviolet B radiation. (2) Apoptosis, reactive oxygen species content, and malondialdehyde levels were increased when Prdx6 was downregulated. This phenomenon became more severe under ultraviolet B radiation. (3) The expression levels of apoptosis-related and antioxidant genes all changed along with the changes in expression of Prdx6. CONCLUSION: (1) Both peroxidase and phospholipase A2 activities of Prdx6 are crucial for its protective role in corneal tissue. (2) Downregulated expression of Prdx6 resulted in high endoplasmic reticulum stress. (3) Apoptosis in human corneal epithelial cells with downregulated Prdx6 coupled with ultraviolet B radiation was related to the pathways of DNA damage and the death receptor. (4) Low levels of antioxidants are sufficient for maintaining homeostasis in human corneal epithelial cells without external stimuli. Under the condition that Prdx6 was downregulated, human corneal epithelial cells were more sensitive to ultraviolet B radiation.

The Menadione-Mediated WST1 Reduction by Cultured Astrocytes Depends on NQO1 Activity and Cytosolic Glucose Metabolism.

The reduction of water-soluble tetrazolium salts (WSTs) is frequently used to determine the metabolic integrity and the viability of cultured cells. Recently, we have reported that the electron cycler menadione can efficiently connect intracellular oxidation reactions in cultured astrocytes with the extracellular reduction of WST1 and that this menadione cycling reaction involves an enzyme. The enzymatic reaction involved in the menadione-dependent WST1 reduction was found strongly enriched in the cytosolic fraction of cultured astrocytes and is able to efficiently use both NADH and NADPH as electron donors. In addition, the reaction was highly sensitive towards dicoumarol with Kic values in the low nanomolar range, suggesting that the NAD(P)H:quinone oxidoreductase 1 (NQO1) catalyzes the menadione-dependent WST1 reduction in astrocytes. Also, in intact astrocytes, dicoumarol inhibited the menadione-dependent WST1 reduction in a concentration-dependent manner with half-maximal inhibition observed at around 50 nM. Moreover, the menadione-dependent WST1 reduction by viable astrocytes was strongly affected by the availability of glucose. In the absence of glucose only residual WST1 reduction was observed, while a concentration-dependent increase in WST1 reduction was found during a 30 min incubation with maximal WST1 reduction already determined in the presence of 0.5 mM glucose. Mannose could fully replace glucose as substrate for astrocytic WST1 reduction, while other hexoses, lactate and the mitochondrial substrate ß-hydroxybutyrate failed to provide electrons for the cell-dependent WST1 reduction. These results demonstrate that the menadione-mediated WST1 reduction involves cytosolic NQO1 activity and that this process is strongly affected by the availability of glucose as metabolic substrate.

Tetrazolium reduction assays under-report cell death provoked by clinically relevant concentrations of proteasome inhibitors.

High throughput cell viability screening assays often capitalize on the ability of active enzymes or molecules within viable cells to catalyze a quantifiable chemical reaction. The tetrazolium reduction (MTT) assay relies on oxidoreductases to reduce tetrazolium into purple formazan crystals that are solubilized so absorbance reflects viability, while other assays use cellular ATP to catalyze a luminescence-emitting reaction. It is therefore important to know how accurately these assays report cellular responses, as cytotoxic anti-cancer agents promote cell death via a variety of signaling pathways, some of which may alter how these assays work. In this study, we compared the magnitude of cytotoxicity to different cell types provoked by currently used anti-cancer agents, using three different cell viability assays. We found the three assays were consistent in reporting the viability of cells treated with chemotherapy drugs or the BH3 mimetic navitoclax, but the MTT assay underreported the killing capacity of proteasome inhibitors. Additionally, the MTT assay failed to confirm the induction of caspase-mediated cell death by bortezomib at physiologically relevant concentrations, thereby mischaracterizing the mode of cell death. While the cell viability assays used allow for the rapid identification of novel cytotoxic compounds, our study emphasizes the importance for these screening assays to be complemented with a direct measure of cell death or another independent measure of cell viability. We caution researchers against using MTT assays for monitoring cytotoxicity induced by proteasome inhibitors.

INT reduction is a valid proxy for eukaryotic plankton respiration despite the inherent toxicity of INT and differences in cell wall structure.

The reduction of 2-para (iodophenyl)-3(nitrophenyl)-5(phenyl) tetrazolium chloride (INT) is increasingly being used as an indirect method to measure plankton respiration. Its greater sensitivity and shorter incubation time compared to the standard method of measuring the decrease in dissolved oxygen concentration, allows the determination of total and size-fractionated plankton respiration with higher precision and temporal resolution. However, there are still concerns as to the method's applicability due to the toxicity of INT and the potential differential effect of plankton cell wall composition on the diffusion of INT into the cell, and therefore on the rate of INT reduction. Working with cultures of 5 marine plankton (Thalassiosira pseudonana CCMP1080/5, Emiliania huxleyi RCC1217, Pleurochrysis carterae PLY-406, Scrippsiella sp. RCC1720 and Oxyrrhis marina CCMP1133/5) which have different cell wall compositions (silica frustule, presence/absence of calcite and cellulose plates), we demonstrate that INT does not have a toxic effect on oxygen consumption at short incubation times. There was no difference in the oxygen consumption of a culture to which INT had been added and that of a replicate culture without INT, for periods of time ranging from 1 to 7 hours. For four of the cultures (T. pseudonana CCMP1080/5, P. carterae PLY-406, E. huxleyi RCC1217, and O. marina CCMP1133/5) the log of the rates of dissolved oxygen consumption were linearly related to the log of the rates of INT reduction, and there was no significant difference between the regression lines for each culture (ANCOVA test, F = 1.696, df = 3, p = 0.18). Thus, INT reduction is not affected by the structure of the plankton cell wall and a single INT reduction to oxygen consumption conversion equation is appropriate for this range of eukaryotic plankton. These results further support the use of the INT technique as a valid proxy for marine plankton respiration.

Toxoplasma gondii induces extracellular traps release in cat neutrophils.

Neutrophils respond differently to violations of the body's physiological barriers during infections. Extracellular traps comprise one of the mechanisms used by these cells to reduce the spread of pathogens to neighboring tissues, as well as ensure a high concentration of antimicrobial agents at the site of infection. To date, this innate defense mechanism has not been previously demonstrated in neutrophils of cats exposed to Toxoplasma gondii. The aim of this study was to characterize the in vitro release of neutrophil extracellular traps (NETs) when neutrophils isolated from cats were exposed to T. gondii. First, cellular viability was tested at different time points after parasite exposure. The production of reactive oxygen species (ROS) and lactate dehydrogenase and the amount of extracellular DNA were quantified. In addition, the number of parasites associated with neutrophils was determined, and the observed NETs formed were microscopically characterized. Results showed that (i) in culture, neutrophils isolated from cats presented diminished cellular viability after 4 h of incubation, and when neutrophils were incubated with T. gondii, they displayed cytotoxic effects after 3 h of interaction; (ii) neutrophils were able to release structures composed of DNA and histones, characterized as NETs under optical, immunofluorescence, and electron scanning microscopy, when stimulated with T. gondii; (iii) only 11.4% of neutrophils were able to discharge NETs during 3 h of incubation; however, it was observed through extracellular quantification of DNA that this small number of cells were able to display different behavior compared to a negative control (no parasite) group; (iv) significant differences in ROS production were observed in neutrophils exposed to T. gondii. In conclusion, our results showed that neutrophils isolated from cats exposed to T. gondii release structures composed of DNA and histones, similar to what has already been described in other neutrophil species infected with the parasite.

Effect of growth media on the MTT colorimetric assay in bacteria.

Reduction of tetrazolium salts to colored formazan products by metabolically active cells is widely used for assessment of cell viability. Among the tetrazolium compounds most commonly used is MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Numerous studies about sites and mechanisms of cellular reduction of MTT, performed in mammalian cell cultures, have identified various parameters that affect formazan production and can lead to overestimation/underestimation of viable cells or effects of treatment. Irrespective of lack of such data for prokaryotic cells, the MTT assay is commonly used for microbiological studies, which often leads to contradictory results or misinterpretation of data. The aim of this study was to investigate how components of growth media and conditions of growth, affect formazan formation by microbial cells. Results showed that MTT reduction depended on the amino acid composition of the medium. Several amino acids potentiated formazan production by Gram-positive and Gram-negative bacteria, with histidine having the strongest effect. Results of this study demonstrate that data obtained with the MTT test should be interpreted with caution, particularly when different growth media are used or treatments affect metabolic pathways, and that evaluation of the reliability of the MTT assay under specific conditions should be performed, to avoid erroneous results. Performing the assay with cells suspend in glucose-supplemented buffer would eliminate the effects of metabolites and will limit cell division during incubation with MTT. Another critical element to be considered is the choice of a proper solvent for dissolution of formazan crystals.

Longitudinal investigation of ischemic stroke using magnetic resonance imaging: Animal model.

BACKGROUND: Arterial embolism is a major cause of ischemic stroke. Currently, digital subtraction angiography (DSA) is the gold standard in clinical arterial embolization examinations. However, it is invasive and risky. OBJECTIVE: This study aims to longitudinally assess the progression of carotid artery embolism in middle cerebral artery occlusion animal model (MCAO) using magnetic resonance imaging (MRI) techniques. METHODS: Turbo spin echo (TSE), time of flight magnetic resonance angiography (TOF-MRA) and diffusion weighted magnetic resonance imaging (DWI) were used to evaluate the image characteristics of cerebral tissues at 1, 2, 3, 7, 14, 21 and 28 days after MCAO microsurgery on Sprague-Dawley (SD) rats. Quantitative analysis was performed and compared in MCAO hemisphere and contralateral normal hemisphere. Furthermore, pathologic section using triphenyl tetrazolium chloride (TTC) stain was performed as well. RESULTS: TOF-MRA showed carotid signal void in the embolism side, which is evidence of artery occlusion. The used MRI techniques showed that edema gradually dissipated within one week, but there was no significant change afterwards. The time-varying signal intensity of MRI techniques in MCAO hemisphere changed significantly, but there were no significant changes in contralateral normal hemisphere. Cerebral injury was also confirmed by analysis of pathology images. CONCLUSIONS: The MCAO animal model was successfully established on SD rats using the microsurgery to assess arterial embolization of intracranial tissue injury.

GC-MS-Based Metabolomics to Reveal the Protective Effect of Gross Saponins of Tribulus terrestris Fruit against Ischemic Stroke in Rat.

Stroke is one of the most common neurological disorders and seriously threatens human life. Gross saponins of Tribulus terrestris fruit (GSTTF) are used for neuroprotective treatment on convalescents of ischemic stroke. However, the therapeutic effects and mechanisms have not yet well understood, especially from the metabolic perspective. In this study, the protective effect of GSTTF on ischemic stroke in a middle cerebral artery occlusion (MCAO) rat model was investigated by the GC-MS-based metabolomics approach. 2,3,5-triphenyltetrazolium chloride (TTC) staining of brain tissues showed that GSTTF significantly reduced the infarct area after MCAO surgery. Metabolomic profiling showed a series of metabolic perturbation occurs in ischemic stroke compared with sham group. GSTTF can reverse the MCAO-induced serum metabolic deviations by regulating multiple metabolic pathways including fatty acids metabolism, amino acids metabolism, and carbohydrates metabolism. The current study provided a useful approach for understanding the mechanism of MCAO-induced ischemic stroke and a reliable basis for evaluating the efficacy of GSTTF in the treatment of ischemic stroke.

Left-right asymmetry influenced the infarct volume and neurological dysfunction following focal middle cerebral artery occlusion in rats.

OBJECTIVE: To investigate the differential effects of left versus right cerebral hemisphere on the infarct volume and behavioral function following focal cerebral ischemia in rats. METHODS AND MATERIALS: Middle cerebral artery occlusion (MCAO) was induced in the right-handed rats by filament insertion for 1.5 hr, and then reperfusion was established according to Zea-Longa method. A total of 36 male Sprague Dawley rats were randomly divided into a left MCAO group or a right MCAO group. The modified neurological severity scores (mNSS), tapered beam-walking test, and Morris water maze experiment were all carried out to evaluate the sensorimotor and cognitive outcomes at the 1d, 3d, and 7d after MCAO, respectively. Infarct volume of the brains was measured by triphenyltetrazolium chloride (TTC) staining. RESULTS: The sensorimotor function was more worse in the left MCAO group than that in the right MCAO group at the 1d, 3d, and 7d after MCAO (p < 0.05). While the cognitive function was much better in the left MCAO group than that in the right MCAO group at the 1d and 3d after MCAO (p < 0.05). But no significant difference was achieved in cognitive function between the two groups at 7d after MCAO (p > 0.05). There was no significant difference in total infarct volume between the two groups at the 1d, 3d, and 7d after MCAO, respectively (p > 0.05). CONCLUSION: The infarct volume is not affected significantly by the left or right MCAO model in the early days. The lesions in the left hemisphere produce more severe sensorimotor impairments, while more severe cognitive impairments are produced by the right hemispherical lesions. These findings suggest that it is structural and functional asymmetry between the two hemispheres other than infarct volume that affects the outcomes of rat MCAO.

Colorimetric assay for active biomass quantification of Fusarium fujikuroi.

A colorimetric assay has been developed for quantitative analysis of active biomass of Fusarium fujikuroi, based on the reduction of the tetrazolium salt 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) when menadione was present as an electron coupling agent. The optimum assay-conditions were set as 200 µg/ml XTT, 5 µM menadione and one-hour reaction time. Under these settings, the produced formazan displayed a linear relationship with F. fujikuroi biomass. This method was subsequently applied to evaluate the cell growth behavior, which showed a positive correlation with the carbon source consumption and gibberellin biosynthesis under the industrial fermentation conditions. Our results showed that the XTT-menadione assay is a valuable tool in analyzing the industrial fermentation process of F. fujikuroi, especially when the medium contains insoluble and complex components.

Cellular glucose metabolism is essential for the reduction of cell-impermeable water-soluble tetrazolium (WST) dyes.

Water-soluble tetrazolium (WST) dyes, such as WST-1 and WST-8, are widely used in cell proliferation and anti-cell-growth drug screen assays. However, the underlying determinants for WST reduction are still largely unknown. In addition, application of tetrazolium-based assays to cellular glucose metabolism studies has not been fully explored. In the present study, we show here that WST-8 reduction is dependent on cellular glucose metabolism. In order to minimize the variance of live cell number during stimulation, we treated cells with different stimuli and performed tetrazolium-based assays within 6 hours. Withdrawal of medium glucose supply greatly attenuated WST-8 reduction but not intracellular ATP levels, while re-adding glucose reconstituted WST-8 reduction, indicating the effect was not due to cell death. The role of glucose on WST-8 reduction is specific since glutamine, fructose or galactose did not substitute for the effect of glucose on WST-8 reduction. Furthermore, inhibition of glucose transporters, intracellular glucose metabolic enzymes or EGFR-PI3K-Akt signaling also attenuated WST-8 reduction. In an attempt to screen inhibitors targeting cellular glucose metabolism from hyperglycemia-associated drugs, it turned out that HIV protease inhibitor, ritonavir, could largely block WST-8 reduction, but not cellular ATP level. Interestingly, ritonavir has been shown to acutely block glucose transport in vitro and in vivo. Taken together, our studies not only demonstrate an essential role of cellular glucose metabolism on WST-8 reduction, but also propose a novel application of tetrazolium-based assays in screening for inhibitors of cellular glucose metabolism when used in combination with ATP assay.

Analysis of Cell Viability by the MTT Assay.

Among viability assays that depend on the conversion of substrate to chromogenic product by live cells, the MTT assay is still among one of the most versatile and popular assays. The MTT assay involves the conversion of the water-soluble yellow dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to an insoluble purple formazan by the action of mitochondrial reductase. Formazan is then solubilized and the concentration determined by optical density at 570 nm. The result is a sensitive assay with excellent linearity up to ∼106 cells per well. As with the alamarBlue assay, small changes in metabolic activity can generate large changes in MTT, allowing one to detect cell stress upon exposure to a toxic agent in the absence of direct cell death. The assay has been standardized for adherent or nonadherent cells grown in multiple wells. The protocol uses a standard 96-well plate. This can be scaled up, however, to suit a different plate format. Plate 500-10,000 cells per well in a 96-well plate. The assay has good linearity up to 106 cells.

Enhanced cell growth on 3D graphene scaffolds implanted with nitrogen ions.

One of the key challenges in engineering tissues for cell-based therapies is developing biocompatible scaffold materials to direct cell behavior. In this paper, the cytocompatibilities of a flexible three-dimensional graphene scaffold (3D-G) and the same scaffold implanted with nitrogen ions (N+/3D-G) are compared using an in vitro assay based on 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The N+/3D-G samples were prepared from low-temperature hydrothermally synthesized flexible 3D-G by ion implantation and were found to display improved adhesion and proliferation of rat osteoblast and mouse fibroblast cells. In particular, the N+/3D-G sample with a nitrogen content of ∼10% showed the highest levels of cell viability and proliferation. The flexible N+/3D-G has potential applications as a biocompatible scaffold material that provides improved surface area and hydrophilic groups for cell growth and proliferation.

The optimal ethanol lock therapy regimen for treatment of biofilm-associated catheter infections: an in-vitro study.

BACKGROUND: Ethanol-based lock therapy (LT) solutions are used as an alternative to antibiotics for the conservative management of catheter-related bloodstream infection. However, no clear consensus on regimen or dose has been reached. AIM: To find the ethanol-based lock solution containing a sufficiently low concentration of ethanol for reduction of the metabolic activity of bacterial and fungal biofilms. METHODS: Using an in-vitro model, three concentrations of ethanol (25%, 40%, 70%) were tested, with and without 60 IU of heparin, at six different time-points and against 24 h preformed biofilms of Staphylococcus aureus ATCC29213, Staphylococcus epidermidis (clinical isolate), Enterococcus faecalis ATCC33186, Candida albicans ATCC14058, and Escherichia coli ATCC25922. The reduction in the metabolic activity of the biofilm was measured using the tetrazolium salt assay and LT was considered to be successful when metabolic activity fell by >90%. Regrowth inhibition was then tested within 24 h and seven days after each LT regimen only at the ethanol concentration of the most successful LT regimen. FINDINGS: The most successful LT was achieved with 40% ethanol + 60 IU of heparin only at 24, 72, and 24 h for seven-day regimens (P < 0.05). However, none of the regimens reached 45% RI within seven days of therapy. CONCLUSION: According to our in-vitro data, an ethanol-based lock solution with 40% ethanol + 60 IU heparin administered daily for 72 h is sufficient to almost eradicate the metabolic activity of bacterial and fungal biofilms. Future studies are needed to study cell regrowth after LT.

Anti-Candida activity of antidepressants sertraline and fluoxetine: effect upon pre-formed biofilms.

As an opportunistic fungal pathogen Candida spp. has the ability to form biofilms. The most prescribed drugs for Candida infections, azoles, have shown to be less effective when biofilms are present. In addition, increasing treatment costs and the fact that most prescribed antifungal drugs have only fungistatic activity justify the search for new treatment strategies. One promising approach is third generation antidepressants, selective serotonin re-uptake inhibitors (SSRIs), because of their proven antifungal activity against several Candida spp. Thus, the aim of this work was to determine the ability of two commonly used SSRIs, fluoxetine and sertraline, to impair both biofilm metabolic viability and biofilm biomass. The in vitro effect of fluoxetine and sertraline was individually tested against biofilm metabolic viability and biofilm biomass using the MTT assay and the Crystal Violet assay, respectively. For both drugs, a dose-dependent reduction on both biofilm metabolism and biomass was present. At high concentrations, fluoxetine was able to reduce biofilm metabolism by 96% (C. krusei) and biofilm biomass by 82% (C. glabrata), when compared to the control. At similar conditions, sertraline achieved a reduction of 88% on biofilm biomass (C. glabrata) and 90% on biofilm metabolism (C. parapsilosis). Moreover, fluoxetine showed interesting anti-biofilm activity at previously reported planktonic MIC values and even at sub-MIC values. These results reinforce the potential interest of SSRIs as anti-biofilm agents to be study to counteract resistance phenomena on candidosis.

Determination of the Selection Capacity of Antibiotics for Gene Selection.

Choosing a potent selection antibiotic (SA), is a crucial success factor when creating stably transfected cell lines using an antibiotic selection marker. The selection capacity of this antibiotic is defined as its ability to kill sensitive, untransfected parental cells, while leaving resistant, transfected cells unharmed. Currently, no procedure has been described to determine this selection capacity. Therefore, a protocol to obtain a numerical value, called the "selectivity factor" (SF), that defines the selection capacity of SAs is developed. The SF is determined by using a modified MTT (3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide) assay for both sensitive and resistant cells, and applies to commonly used cell lines. To prove the concept, the SF of the SA G418 and hygromycin B (HmB) on several cell lines is determined. The SF of G418 on BHK-21 cells is very high, indicating that G418 is an ideal SA for transfected BHK-21 cells. For HeLa cells, the SF of G418 is very low suggesting G418 is not an optimal SA for selecting transfected HeLa cells. For these cells, HmB would be a better choice. These conclusions are confirmed by an independent cell death assay. The SF identifies the most optimal SA for a certain cell line, reduces the risk of selecting spontaneously resistant cell clones, and streamlines the process of generating stable cell lines. Most importantly, the method is especially time saving when obtaining stable cell lines expressing toxic genes, and reduces culture times for generating large numbers of cell lines from the same parental cell line.