Microplate Fluorometer Research Articles

Liquid fluorophore taggants for mark‐release‐recapture research: a survey of potential arthropod targets

AbstractWe evaluated a method for marking arthropods that could serve as a valuable tool for mark‐release‐recapture dispersal research. The taggants tested consisted of three liquid fluorophores labeled cartax green, magenta, and orange. The manufacturer markets these fluorescent markers as forensic theft deterrents. Specimens of 16 genera of arthropods were externally marked with either one of the colored fluorophores or with water (negative control treatment). The specimens were then qualitatively and quantitatively inspected for fluorescence 24 h later. For the qualitative analysis, three independent observers scored each specimen by direct observation for the presence of a fluorescent mark. The specimens were scored using a portable ultraviolet (UV) tube lantern and a specialized NIGHTSEA‐brand LED UV light. The three fluorophores were readily detected on many but not all the species examined, regardless of the type of UV light used. Moreover, the NIGHTSEA LED light yielded fewer false‐negative observer errors than the lantern. Each specimen's fluorescence was measured with an automated dual‐wavelength microplate fluorometer for the quantitative analysis. Overall, the quantitative analysis was very reliable at detecting fluorescence on a few taxa [e.g., Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), Chrysoperla spp. (Neuroptera: Chrysopidae)], effective on most taxa, and unreliable on several others [e.g., Collops vittatus (Say) (Coleoptera: Melyridae), Geocoris spp. (Hemiptera: Geocoridae), Mecaphesa celer (Hentz) (Araneae: Thomisidae)]. The cartax green marker was more readily detected than the magenta and orange markers with both visual and automated detection. Overall, the results show that these fluorophores could be effective markers for many arthropod species.

Use of a Fluorophore to Tag Arthropods for Mark-Release-Recapture Type Research.

We examined the feasibility of externally marking insects with the liquid fluorescent forensic theft deterrent, SmartWater (SmartWater CSI, LLC.). We sprayed captive Lygus hesperus (Knight) (Hemiptera: Miridae), Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), and Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae) with SmartWater fluorophore, and then qualitatively examined them for fluorescence by visual inspection under ultraviolet (UV) light and quantitatively measured them with a multiwavelength microplate fluorometer. The results indicate that this product has enormous potential as a taggant for L. hesperus and B. tabaci. However, the marking efficiency for H. convergens was only adequate. The advantages and limitations of using SmartWater as a biological marker for arthropod mark-release-recapture research are discussed.

QDNase assay: A quantitative method for real-time assessment of DNase activity on coated surfaces

DNase coatings show great potential to prevent biofilm formation in various applications of the medical implant, food and marine industry. However, straightforward and quantitative methods to characterize the enzymatic activity of these coatings are currently not available. We here introduce the qDNase assay, a quantitative, real-time method to characterize the activity of DNase coatings. The assay combines (1) the use of an oligonucleotide probe, which fluoresces upon cleavage by coated DNases, and (2) the continuous read-out of the fluorescent signal within a microplate fluorometer format. The combination of these two properties results in a real-time fluorescent signal that is used to directly quantify the activity of DNase coatings. As a proof of concept, bovine DNase I coatings were immobilized on titanium by means of chemical grafting and their activity was estimated at 3.87 × 10-4 U. To our knowledge, the qDNase assay provides the first approach to report the activity of a DNase coating in absolute DNase activity units. This assay will not only serve to compare existing DNase coating methods more accurately, but will also enable the rational design of new DNase coating methods in the future.

Rapid screening fluorescence method applied to detection and quantitation of paralytic shellfish toxins in invertebrate marine vectors

ABSTRACTA rapid screening method is described for the determination of paralytic shellfish toxins (PST), in fresh marine vectors (bivalves and gastropods), at levels ranging from 0.05 to 5.0 mg STX-eq kg−1. PST are extracted from marine vector homogenates with acetic acid according to the Pre-COX-LC-FLD method. At the same time, the obtained extract is oxidised simultaneously in hydrogen peroxide and periodate oxidate to determine PST, non-N-hydroxylated and N-hydroxylated toxins, respectively. Then, they are analysed using a microplate fluorometer (Ex: 335 nm/Em: 405 nm). All the samples were compared with the liquid chromatography post-column oxidation method. Recoveries of PST added to fresh and processed marine vectors averaged 93.9% with a coefficient of variation of 6.1%. Both methods showed a good linear regression (r2 = 0.97). The method shows good intra- and inter-day precisions with a relative coefficient of variation of ≈ 3.8% and 5.7%, respectively. The limit of quantification of the rapid screening fluorescence method was ≈ 0.082 mg STX-eq kg−1, with ≤5% false positives. The established rapid screening fluorescence methods offer highly effective and verifiable pre-analyses of PST contamination in marine vectors and can be used for routine screening of the PST in seafood before formal identification by confirmatory methods (Pre-COX LC-FLD method, Lawrence method).

ANTIHYPERTENSIVE ACTIVITY OF QUINOA (Chenopodium quinoa Willd.) PROTEIN HYDROLYSATES

Background: Nowadays, there has been an increase in the number of studies focused on the search for bioactive
 compounds produced by hydrolytic reactions from natural sources, such as the Quinoa grain, which represents an
 interesting agro-alimentary source that can have a beneficial influence on health, specifically antihypertensive potential. For
 this reason, the aim of the present study was to evaluate the antihypertensive activity of the protein hydrolysates obtained of
 Quinoa, which results important at the time to consider the incorporation of such peptides in the design of functional foods.
 Materials and Methods: Quinoa (Chenopodium quinoa Willd.) seeds were ground and the obtained flour was degreased
 and the protein isolate was obtained by isoelectric precipitation. The protein isolate was enzymatically hydrolyzed with
 Alcalase® and Flavourzyme® and the antihypertensive effect of peptides against angiotensin converting enzyme was
 evaluate using a mixture of 50 μL of sample, 50 μL of ACE working solution, 200 μL of substrate working solution and the
 fluorescence was determined with a microplate fluorometer following these characteristics: λ (excitation) = 355-375 nm; λ
 (emission) = 400-430 nm.
 Results: Peptides obtained using Alcalase® (protein content= 72.13%; DH= 31.22%) showed the highest inhibitory
 activity against the angiotensin converting enzyme (ACE), close to 88%.
 Conclusion: The Quinoa protein hydrolysates can be considered as a new agri-food source to be incorporated in the
 elaboration of functional foods with antihypertensive potential.

Possible effect of N-Acetyl-L-cysteine on Aβ(25-35)-induced tau hyperphosphorylation

To investigate the possible effect and mechanism of N-Acetyl-L-cysteine (NAC) on fibrillar Aβ(25-35)-induced tau hyperphosphorylation. The phosphorylation of tau was induced by Aβ(25-35) in primary cortical neuron. Neurons were incubated in the absent or present Aβ(25-35), or pre-incubated NAC then co-incubated in Aβ. The measurement of ROS was performed on a microplate fluorometer. The proteins of p35/p25, cdk5, pT205 and pS404 were detected by Western blot. In Aβ treated group, the ROS, pT205 and pS404 level were obviously higher than that in non-treated with Aβ group for 12 h (t=-6.35, P<0.05; t=-5, P<0.05; t=-4.57, P<0.05). However, in neurons pre-incubated with (10 mmol/L) NAC and then co-incubated with 20 µmol/L Aβ, the ROS, pT205 and pS404 level were significantly decreased compared with that of Aβ group (t=3.47, P<0.05; t=3.88, P<0.05 and t=3.64, P<0.05); Upon Aβ exposure for 12 h, cortical neurons showed a statistically significant increase in p25 when compared to the control group (t=-6.20, P<0.05). However, pre-treatment with NAC showed a decrease in p25 as compared to neurons treated with Aβ alone for 12 h (t=4.72, P<0.05). NAC attenuated the Aβ(25-35)-induced tau hyperphosphorylation through CDK5 pathway.

Electrical Pulse Stimulation Induced Changes On Lipid, Mitochondrial, Glut4 And Ampk Content In Human Myotubes

It has been previously shown that human myotubes retain certain in vivo characteristics of the donors. Furthermore, we have shown that Electrical pulse stimulation (EPS), an in vitro exercise mimetic, increases mitochondrial and lipid content in cultured human myotubes after 24 hr. of stimulation. PURPOSE: We aimed to examine the EPS induced adaptations to lipid, mitochondrial, Glucose transporter 4 (GLUT4) and total 5’ Adenosine Monophosphate Activated Protein Kinase (AMPK) content using human myotubes from lean donors. Additionally, we investigated the impact of cell harvest time (immediate vs. 24 hr. after the EPS cessation) on the above-mentioned parameters. METHODS: EPS was applied to myotubes for 60 min (bipolar pulses of 100 Hz, 30V, every 5 seconds), 24 hr. or 48 hr. (single bipolar pulses of 1 Hz for 2 ms; 30V) and were harvested at two different time points: immediately after (early harvest) and 24hr after (late harvest) the end of stimulation. Mitochondrial and lipid content were measured by immunohistochemistry using primary antibody for complex IV and bodipy green dye, respectively. A microplate fluorometer was used to quantify fluorescence signals. GLUT4 and AMPK were measured using western immunoblotting. RESULTS: Lipid in myotubes was significantly higher (p<0.05) after 24 hr early harvest EPS compared to unstimulated control. There was no significant changes in lipid content with late harvest EPS. Mitochondrial content was significantly higher (p<0.05) after acute, 24 hr. as well as 48 hr. early harvest EPS, but was significantly lower (p<0.05) after acute late harvest EPS, compared to unstimulated controls. GLUT4 protein content was higher after acute and 24 hr. early harvest and 48 hr. late harvest EPS. Total AMPK was higher after 24 hr. early harvest and 48 hr. late harvest. AMPK was lower after 24 hr. late harvest. CONCLUSION: These findings suggest 24 hours of EPS treatment lead to increase in lipid, mitochondria, GLUT4 and AMPK protein content in human myotubes cultured from lean donors. Additionally, cell-harvesting time a reflection of post-exercise recovery is a key factor for these adaptations.

Time-Dependent Effects of Anesthetic Isoflurane on Reactive Oxygen Species Levels in HEK-293 Cells.

The inhalation anesthetic isoflurane has been reported to induce caspase activation and apoptosis, which may lead to learning and memory impairment. However, the underlying mechanisms of these effects are largely unknown. Isoflurane has been shown to induce elevation of cytosol calcium levels, accumulation of reactive oxygen species (ROS), opening of the mitochondrial permeability transition pore, reduction in mitochondria membrane potential, and release of cytochrome c. The time course of these effects, however, remains to be determined. Therefore, we performed a pilot study to determine the effects of treatment with isoflurane for various times on ROS levels in HEK-293 cells. The cells were treated with 2% isoflurane plus 21% O2 and 5% CO2 for 15, 30, 60, or 90 min. We then used fluorescence imaging and microplate fluorometer to detect ROS levels. We show that 2% isoflurane for 60 or 90 min, but not 15 or 30 min, induced ROS accumulation in the cells. These data illustrated that isoflurane could cause time-dependent effects on ROS levels. These findings have established a system to further determine the time course effects of isoflurane on cellular and mitochondria function. Ultimately, the studies would elucidate, at least partially, the underlying mechanisms of isoflurane-induced cellular toxicity.

The Impact of Storage Temperature on the Morphology, Viability, Cell Number and Metabolism of Cultured Human Conjunctival Epithelium

Purpose: To evaluate the effect of storage temperature on the morphology, viability, cell number and metabolism of cultured human conjunctival epithelial cells (HCjEs).Materials and Methods: Three-day cultured HCjEs were stored at nine different temperatures between 4 °C and 37 °C for four and seven days. Phenotype was assessed by immunofluorescence microscopy, morphology by scanning electron microscopy, viability and cell number by a microplate fluorometer and glucose metabolism by a blood gas analyzer.Results: Cultured cells not subjected to storage expressed the conjunctival cytokeratins 7 and 19 and the proliferation marker proliferating cell nuclear antigen. Cell morphology was best maintained following four-day storage between 12 °C and 28 °C and following 12 °C storage after seven days. Assessed by propidium iodide uptake, the percentage of viable cells after four-day storage was maintained only between 12 °C and 28 °C, whereas it had decreased in all other groups (p < 0.05; n = 4). After seven days this percentage was maintained in the 12 °C group, but it had decreased in all other groups, compared to the control (p < 0.05; n = 4). The total number of cells remaining in the cultures after four-day storage, compared to the control, had declined in all groups (p < 0.05; n = 4), except 12 °C and 20 °C groups. Following seven days this number had decreased in all groups (p < 0.01; n = 4), except 12 °C storage. Four-day storage at 12 °C demonstrated superior preservation of the number of calcein-stained viable cells (p < 0.05) and the least accumulation of ethidium homodimer 1–stained dead cells (p < 0.001), compared to storage at 4 °C and 24 °C (n = 6). The total metabolism of glucose to lactate after four-day storage was higher in the 24 °C group compared to 4 °C and 12 °C groups, as well as the control (p < 0.001; n = 3).Conclusions: Storage at 12 °C appears optimal for preserving the morphology, viability and total cell number in stored HCjE cultures. The superior cell preservation at 12 °C may be related to temperature-associated effects on cell metabolism.

Antioxidants Improve the Viability of Stored Adult Retinal Pigment Epithelial-19 Cultures.

IntroductionThere is increasing evidence that retinal pigment epithelium (RPE) can be used to treat age-related macular degeneration, one of the leading causes of blindness worldwide. However, the best way to store RPE to enable worldwide distribution is unknown. We investigated the effects of supplementing our previously published storage method with seven additives, attempting to improve the number of viable adult retinal pigment epithelial (ARPE)-19 cells after storage.Materials and methodsARPE-19 cells were cultured on multiwell plates before being stored for 1 week at 16 °C. Unsupplemented Minimal Essential Medium (MEM) (control) and a total of seven individual additives (DADLE ([d-Ala2, d-Leu5]-encephalin), capsazepine, docosahexaenoic acid (DHA), resveratrol, quercetin, simvastatin and sulforaphane) at three to four concentrations in MEM were tested. The individual effect of each additive on cell viability was analyzed with a microplate fluorometer. Cell phenotype was investigated by both microplate fluorometer and epifluorescence microscopy, and morphology by scanning electron microscopy.ResultsSupplementation of the storage medium with DADLE, capsazepine, DHA or resveratrol significantly increased the number of viable cells by 86.1% ± 41.9%, 67.9% ± 24.7%, 36.5% ± 10.3% and 21.1% ± 6.4%, respectively, compared to cells stored in unsupplemented MEM. DHA and resveratrol significantly reduced caspase-3 expression, while expression of RPE65 was maintained across groups.ConclusionThe number of viable ARPE-19 cells can be increased by the addition of DADLE, capsazepine, DHA or resveratrol to the storage medium without perturbing apoptosis or differentiation.Electronic supplementary materialThe online version of this article (doi:10.1007/s40123-014-0024-9) contains supplementary material, which is available to authorized users.

Panax ginseng induces the expression of CatSper genes and sperm hyperactivation.

The cation channel of sperm (CatSper) protein family plays important roles in male reproduction and infertility. The four members of this family are expressed exclusively in the testis and are localized differently in sperm. To investigate the effects of Panax ginseng treatment on the expression of CatSper genes and sperm hyperactivation in male mice, sperm motility and CatSper gene expression were assessed using a computer-assisted semen analysis system, a Fluoroskan Ascent microplate fluorometer to assess Ca2+ influx, real-time polymerase chain reaction, Western blotting and immunofluorescence. The results suggested that the Ca2+ levels of sperm cells treated with P. ginseng were increased significantly compared with the normal group. The P. ginseng-treated groups showed increased sperm motility parameters, such as the curvilinear velocity and amplitude of lateral head displacement. Taken together, the data suggest that CatSper messenger ribonucleic acid levels were increased significantly in mouse testes in the P. ginseng-treated group, as was the protein level, with the exception of CatSper2. In conclusion, P. ginseng plays an important role in improving sperm hyperactivation via CatSper gene expression.

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Role of the mannose receptor in phagocytosis of Enterococcus faecalis strain EC‐12 by antigen‐presenting cells

The aim of this study was to clarify the phagocytic mechanisms of a heat-killed cell preparation of Enterococcus faecalis strain EC-12 (EC-12) by antigen-presenting cells (APCs). Fluorescein isothiocyanate (FITC)-labeled EC-12 was cocultured with peritoneal macrophage and the amount of EC-12 phagocytosed by peritoneal macrophages was measured using a microplate fluorometer. Peritoneal macrophages from toll-like receptor (TLR)2-, TLR7-, and MyD88-deficient knockout (KO) mice exhibited similar levels of EC-12 phagocytosis to those from wild-type mice. Similarly, dectin-1 neutralization of peritoneal macrophages had no effect on EC-12 phagocytosis. However, blockade of the mannose receptor (MR) significantly decreased the amount of EC-12 phagocytosed by peritoneal macrophages; the same effect was observed in bone marrow-derived macrophages and dendritic cells. Our findings suggest that MR plays a major role in EC-12 phagocytosis by the APCs.This aim of this study was to clarify the phagocytic mechanisms of a heat-killed cell preparation of Enterococcus faecalis strain EC-12 (EC-12) by antigen-presenting cells (APCs). Our findings suggest that mannose receptor (MR) plays a major role in EC-12 phagocytosis by the APCs.

Abstract 1953: Rapid quantitation of circulating microRNAs using a novel detection technology.

Abstract The rapid implementation of quantifying microRNAs (miRNAs) in body fluids for diagnosis and prognosis could be improved by designing more rapid approaches. The current mainstream approach is quantitative reverse transcriptase PCR (qRT-PCR). Barriers for the applicability are lengthy time requirement for isolation and amplification of miRNAs, and polymerase inhibitors that copurify with miRNA, and compromise the accuracy of the approach. Here we provide an approach using a nano-particle technology that overcomes these limitations. This technology is based on the principle that a fluorophore-containing oligonucletide sequence is fluorescently silent unless displaced from a quencher by a specific substrate RNA. Once displacement occurs, the resulting fluorescence provides a measure of RNA concentration in the reaction. Our approach allows for the direct and rapid quantification of circulating miRNAs with minimal processing of blood plasma/serum and is done without the use of enzymes. Using circulating miRNAs with established roles in cancer and quality control, miR-16, miR-21 and miR-22 and miR-451, we identified optimal temperature, detergent and solvent-extraction parameters, to quantify accurately these miRNAs in plasma using a microplate fluorometer within an hour after plasma preparation. The stringency of our conditions can discriminate wild type from a single-basepair mismatch RNA, and is specific for mature miRNAs. Furthermore, this approach facilitates measurement of samples collected in heparin, an anticoagulant that severely inhibits polymerases used for qRT-PCR. This approach creates the possibility for rapid blood tests to quantify miRNA-associated events in real-time, and provides greater public access to miRNA studies by reducing the cost and time requirement of current accurate approaches of miRNA quantitation. Citation Format: Dominik Duelli, Jaime Palma, Don Weldon, Justin Doong, Grace Johnston, Fellars Stacey, Michelle Hastings. Rapid quantitation of circulating microRNAs using a novel detection technology. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1953. doi:10.1158/1538-7445.AM2013-1953

Optimization of Storage Temperature for Cultured ARPE-19 Cells

Purpose. The establishment of future retinal pigment epithelium (RPE) replacement therapy is partly dependent on the availability of tissue-engineered RPE cells, which may be enhanced by the development of suitable storage methods for RPE. This study investigates the effect of different storage temperatures on the viability, morphology, and phenotype of cultured RPE. Methods. ARPE-19 cells were cultured under standard conditions and stored in HEPES-buffered MEM at nine temperatures (4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C, and 37°C) for seven days. Viability and phenotype were assessed by a microplate fluorometer and epifluorescence microscopy, while morphology was analyzed by scanning electron microscopy. Results. The percentage of viable cells preserved after storage was highest in the 16°C group (48.7% ± 9.8%; P < 0.01 compared to 4°C, 8°C, and 24°C–37°C; P < 0.05 compared to 12°C). Ultrastructure was best preserved at 12°C, 16°C, and 20°C. Expression of actin, ZO-1, PCNA, caspase-3, and RPE65 was maintained after storage at 16°C compared to control cells that were not stored. Conclusion. Out of nine temperatures tested between 4°C and 37°C, storage at 12°C, 16°C, and 20°C was optimal for maintenance of RPE cell viability, morphology, and phenotype. The preservation of RPE cells is critically dependent on storage temperature.

Regulation of the Proteasome by Neuronal Activity and Calcium/Calmodulin-dependent Protein Kinase II

Protein degradation via the ubiquitin proteasome system has been shown to regulate changes in synaptic strength that underlie multiple forms of synaptic plasticity. It is plausible, therefore, that the ubiquitin proteasome system is itself regulated by synaptic activity. By utilizing live-cell imaging strategies we report the rapid and dynamic regulation of the proteasome in hippocampal neurons by synaptic activity. We find that the blockade of action potentials (APs) with tetrodotoxin inhibited the activity of the proteasome, whereas the up-regulation of APs with bicuculline dramatically increased the activity of the proteasome. In addition, the regulation of the proteasome is dependent upon external calcium entry in part through N-methyl-D-aspartate receptors and L-type voltage-gated calcium channels and requires the activity of calcium/calmodulin-dependent protein kinase II (CaMKII). Using in vitro and in vivo assays we find that CaMKII stimulates proteasome activity and directly phosphorylates Rpt6, a subunit of the 19 S (PA700) subcomplex of the 26 S proteasome. Our data provide a novel mechanism whereby CaMKII may regulate the proteasome in neurons to facilitate remodeling of synaptic connections through protein degradation.

Measuring RNA:DNA ratios in individual Acartia tonsa (Copepoda)

Acartia tonsa Dana is a dominant copepod in coastal waters and is therefore an important link in the food web between microplankton and higher trophic levels. RNA:DNA ratios have been used to describe growth and nutritional condition of field-collected copepods and to show strong correlation between RNA:DNA ratios and group egg production (EP). A method was developed using a sensitive, nucleic acid-specific fluorescent dye, and automated microplate fluorometer to measure DNA, RNA, and the RNA:DNA ratio of individual A. tonsa. DNA, RNA, and RNA:DNA ratios and EP were all significantly higher in copepods fed Thalassiosira sp. compared to starved copepods. There was a general trend toward an increase in RNA:DNA ratios with increase in EP, but due to the high degree of variation in both RNA:DNA ratios and EP among individual copepods there was no significant correlation between RNA:DNA ratios and EP. Significant differences in RNA:DNA ratios between fed (7.2) and starved (3.3) copepods were found after 2 days. This assay may be applied to other species of copepods sampled in the field to provide an index of the health of planktonic food webs.

Characterization of methods for determining sterilization efficacy and reuse efficiency of oxygen biosensor multiwell plates

High-throughput screening (HTS) assays based upon fluorometric detection of oxygen consumption in microtiter plates were primarily developed for applications in drug discovery and ecotoxicology but have recently been adopted for use in microbial community-level physiological profiling assays (CLPP). The widespread use of oxygen biosensor systems for CLPP applications has, however, been hindered by the relatively high cost of oxygen biosensor reagent systems and limited access to microplate fluorometer instrumentation platforms. The ability to recycle and reuse oxygen biosensor system plates would expand their utilization for CLPP assays and other research applications in microbial ecology. Here, the efficacy and cost effectiveness of multiple procedures for sterilization of Oxygen Biosensor System (OBS; BD Biosciences) plates for reuse was evaluated. OBS plates were sterilized using ethylene oxide, ultraviolet radiation, and bleach treatments, then evaluated for biosensor response and plate life-cycle performance. Of the sterilization methods tested, ethylene oxide sterilization was most effective based on its low cost, high sterilization efficacy, and minimal impact upon OBS plate response.

A New Polymer–Lipid Hybrid Nanoparticle System Increases Cytotoxicity of Doxorubicin Against Multidrug-Resistant Human Breast Cancer Cells

This work is intended to develop and evaluate a new polymer-lipid hybrid nanoparticle system that can efficiently load and release water-soluble anticancer drug doxorubicin hydrochloride (Dox) and enhance Dox toxicity against multidrug-resistant (MDR) cancer cells. Cationic Dox was complexed with a new soybean-oil-based anionic polymer and dispersed together with a lipid in water to form Dox-loaded solid lipid nanoparticles (Dox-SLNs). Drug loading and release properties were measured spectrophotometrically. The in vitro cytotoxicity of Dox-SLN and the excipients in an MDR human breast cancer cell line (MDA435/LCC6/MDR1) and its wild-type line were evaluated by trypan blue exclusion and clonogenic assays. Cellular uptake and retention of Dox were determined with a microplate fluorometer. Dox-SLNs were prepared with a drug encapsulation efficiency of 60-80% and a particle size range of 80-350 nm. About 50% of the loaded drug was released in the first few hours and an additional 10-20% in 2 weeks. Treatment of the MDR cells with Dox-SLN resulted in over 8-fold increase in cell kill when compared to Dox solution treatment at equivalent doses. The blank SLN and the excipients exhibited little cytotoxicity. The biological activity of the released Dox remained unchanged from fresh, free Dox. Cellular Dox uptake and retention by the MDR cells were both significantly enhanced (p < 0.05) when Dox was delivered in Dox-SLN form. The new polymer-lipid hybrid nanoparticle system is effective for delivery of Dox and enhances its efficacy against MDR breast cancer cells.

Thalidomide increases thrombin generation in multiple myeloma patients

7602 Background: Numerous reports have demonstrated that the clinical use of thalidomide (THAL) carries a risk for deep venous thrombosis. Only limited investigations into the causative mechanisms for this complication have been conducted. Thrombin generation is a global measurement of hyper or hypocoagulability that has not been used to examine the hypercoagulable state caused by THAL. The aim of this study was to determine if THAL affects thrombin generation in patients with multiple myeloma (MM). Methods: MM patients on and off THAL were recruited. Patients were considered to be “on THAL” if they had been taking the medication for at least three months and “off THAL” if they had not been taking the medication for at least three months. Coagulation tubes of blood containing 3.2% sodium citrate were drawn from each patient. Platelet poor plasma was prepared from these specimens using standard technique. The samples were snap frozen at -70 C and stored until analysis. Thrombin generation was measured using the Calibrated Automated Thrombogram as described by Hemker(Pathophysiol Haemo Thromb 2003;33:4–15) using a microplate fluorometer, the Fluoroskan Ascent from Thermo Electron and the Thrombinoscope software package. The endogenous thrombin potential or ETP (area under the thrombin generation curve) and peak thrombin generation (the maximum release of thrombin per unit of time) were the variables of interest for this study. Results: Twenty seven patients were recruited. Five were excluded from analysis because they were on warfarin or enoxaparin. The normal ETP in females is 1803 ± 241 nM*min and in males is 1745 ± 259 nM *min. Normal peak thrombin generation in females is 318.5 ± 52.9 nM and in males is 293.4 ± 48.5 nM. The t-test was used to compare subjects to normal values.Peak thrombin generation was significantly increased in the 9 subjects taking THAL, 374 ± 10.6 nM (p&lt;0.001) and in the 13 subjects not taking THAL, 352 ± 14.7 nM (p=0.009) when compared to normal values. The ETP was not significantly increased 1818 ± 10.7 nM*min (p=0.69) in subjects not taking THAL. However, ETP was increased in subjects taking THAL, 1939± 90 nM*min and showed a trend toward statistical significance (p=0.10). Conclusions: Thalidomide therapy increases thrombin generation significantly in patients with MM. [Table: see text]