ELISA is routinely used for assaying various proteins. The technique has some limitations. The most important limitation is its low sensitivity in detecting ultra-low-concentrated proteins [6, 7]. On the other hand, signal DNA amplification-based methods have several advantages, including easy preparation of nucleic acids and specificity of sequence of signal DNA and its easy amplification [8]. For this reason, it has emerged as a powerful technique, known

as ‘immuno-PCR’ or ‘iPCR’ through introduction of 100 to 10,000 times more sensitivity for detection of target proteins compared with routine ELISA [9]. Although iPCR have been Cabozantinib ic50 designed to detect many proteins [10–19], it may suffer from important limitations including complicated protocol as well as requirement of special instruments and well-trained laboratory personnel. Therefore, it became necessary to design novel techniques to overcome the problems of iPCR [20]. Beyond iPCR other similar techniques have been proposed for detection of protein molecules with ZD1839 nmr DNA as signal molecules. iReal-time PCR, immuno-rolling circle amplification (iRCA), and immuno-nucleic acid sequence-based amplification

(iNASBA) are common examples of such methods. These methods have their own limitations as well, as discussed below. In this study, we propose a new method for protein detection. The proposed method comprises of two main steps, including signal amplification step, called immuno-loop-mediated isothermal amplification (immuno-LAMP or iLAMP), followed by ultra-sensitive detection of amplified signal. Here we discuss the main aspects of this new technique while comparing it with current nucleic acid-based detection methods for proteins. The hypothesis

and its evaluation Immuno-LAMP Loop-mediated isothermal amplification (LAMP) is a new method developed in year 2000 by Notomi et al. Basically, this method of DNA amplification uses a specific DNA polymerase enzyme and a set of four specific primers that distinguish six different regions on the sequence of the target from DNA. The primers consist of inner primer pair [FIP (forward inner primer) and BIP (backward inner primer)] and outer primer pair [F3 (forward outer primer) B3 (backward outer primer)]. Inner primers contain sequences of the sense and antisense strands of the target, while outer primers contain only the antisense sequence of the target strands. In the first step of LAMP, an inner primer starts the reaction and the newly produced strand is displaced by annealing of an outer primer on the same target strand and subsequent synthesis of complementary product strand. The displaced product strand (primed by inner primer) itself serves as template for synthesis of new strand primed by the second inner and outer primers, which hybridize to the other end of the target DNA; the strand adopts stem-loop structure.

Secondly, the coalescence of subsequently ejected ink droplets would cause edges in a type of wave rather than a straight line. Although this phenomenon could be modified by adjusting Apoptosis inhibitor the component of the solvent, the wave-like edge is hard to avoid which would be even worse accompanied with the patterns at the nanometer scale, leading to conduction between the adjacent lines detrimental to the device [21]. Besides, both the low printing speed of inkjet

printing and general time-consuming post sintering process hinder the potential of silver nanoparticle inks for the cost-effective fabrication of printed electronics [22]. Alternatively, emerged as a promising method, spray coating has been successfully applied in printing electronics [23, 24]. Compared to inkjet printing, spray coating exhibits higher ATR inhibitor printing speed and easier control of the deposited film morphology [25]. However, there are only a few reports about spray-coated conductive patterns based on silver nanoparticle inks until now [22, 26]. Therefore, in this work, the influence of spray coating silver nanoparticle inks on the properties of silver nanoscale conductive patterns was studied, and the morphology of the conductive

patterns was characterized and analyzed by scanning electron microscopy (SEM) and electronic dispersive spectrometry (EDS) in detail. Also, based on the obtained silver nanoscale conductive

patterns, polymer solar cells were fabricated using spray coating method, and the performance of the solar cells was also investigated. Methods The device fabrication apparatus is shown in Figure 1a. The silver nanoparticle inks in solution were kept in a bottle and then sprayed directly onto the substrate under the pressure of nitrogen [27]. The shadow Cell Penetrating Peptide mask was utilized for patterning the image on the substrate, which was settled on the heater band for in situ annealing during the spray coating process. For the polymer solar cell (PSC) fabrication, the device configuration is indium tin oxide (ITO)/ZnO (40 nm)/poly(3-hexylthiophene) (P3HT)/ [6]-phenyl-C61-butyric acid methyl ester (PC61BM) (200 ± 15 nm)/PEDOT:PSS (30 nm)/spray-coated Ag [28–30]. ITO-coated glass substrates with a sheet resistance of 10 Ω/sq were consecutively cleaned in an ultrasonic bath containing detergent, acetone, deionized water, and ethanol for 10 min each step and then dried by nitrogen blow. Prior to the deposition of functional layers, the substrate was treated by UV light for 10 min. The ZnO precursor was prepared by dissolving zinc acetate dihydrate (Zn(CH3COO)2 · 2H2O, 99.9%, 1 g, Aldrich, St. Louis, MO, USA) and ethanolamine (NH2CH2CH2OH, 99.5%, 0.28 g, Aldrich) in 2-methoxyethanol (CH3OCH2CH2OH, 99.8%, 10 ml, Aldrich) under vigorous stirring for 12 h for the hydrolysis reaction in air.

Up to now, most of the research on superhydrophobic surface focused on Fer-1 concentration measuring the CAs and sliding angles (SAs) of water droplets with a volume not smaller than 2 μL (approximately 1.6 mm in diameter). However, we often observe water droplets with a volume lower than 2 μL, such as fog droplets, existing or

sliding on a solid surface in nature. There is a need to reveal the interfacial interaction between superhydrophobic surface and tiny water droplets. Generally, pristine carbon nanotubes (CNTs) are hydrophobic materials, which have also been used to construct a superhydrophobic surface [15, 16]. By making micropatterns, the hydrophobicity of a CNT surface is further enhanced. The CA between water and CNT pattern is usually larger than 150°, but the SA is

also large (usually larger than 30°) [17, 18]. However, the superhydrophobic CNT forest might also Idasanutlin price absorb water, resulting in collapsing into cellular foams when water evaporates from interstices of nanotubes [19]. After wetting, the CNT forest might lose its superhydrophobic properties. It needs to construct a stable and durable superhydrophobic surface even wetted by vapor or tiny water droplets. Here, we fabricate the superhydrophobic hierarchical architecture of CNTs on Si micropillar array (CNTs/Si-μp) with large CA and ultralow SA. The CNTs/Si-μp show a durable superhydrophobic surface even after wetting using tiny water droplets. Methods Si micropillar (Si-μp) arrays with defined squares (see Figure  1a, inset) were etched

from a Si (100) wafer by ultraviolet lithography (UVL) and deep reactive-ion etching (DRIE) in sulfur hexafluoride (SF6) and perfluoro-2-butene (C4F8). The height of the Si-μp was controlled by etching time. A standard cleaning process developed by the company Radio Corporation of America (RCA) was carried out to eliminate residual metal and organic species followed by removing Si oxide in a buffered HF solution. The Si micropillar arrays and planar Si wafer were coated with a thin layer of aluminum (10 nm) using an e-beam evaporator for CNT growth. CNTs were grown by floating chemical vapor deposition method, using xylene as carbon source, STK38 ferrocene as catalyst precursor, and a mixture of Ar and H2 as carrier gas, according to our previous report [20]. During the growth of CNTs, the ferrocene/xylene solution (20 mg/mL) was fed into the reactor at a rate of 0.2 mL/min, and Ar and H2 were fed at 400 and 50 sccm, respectively. Figure 1 SEM characterization of various samples. (a) Si micropillar array. (b) Hierarchical architecture of CNTs/Si-μp. (c) Connection between a Si micropillar and CNT forests. (d) CNT forest growing on a planar Si wafer. The samples were characterized using a scanning electron microscope (SEM). The CA and SA were measured using a contact angle goniometer (Rame-hart 300, Rame-hart Instrument Co., Succasunna, NJ, USA).

Though cephalosporins are used as standard treatment, they can be hydrolyzed by β-lactamases at high inocula (‘inoculum effect’), resulting in clinical failures [33–40]. Conventional ASTs typically utilize 5*105 CFU/ml as standard test inoculums [41, 42]. Koing et al. studied the efficacy of several antibiotics against Escherichia coli and S. aureus, and cited much higher bacterial numbers in infections compared to numbers used in standard susceptibility tests as a major reason for predicted antibiotic susceptibility

not matching with observed efficacy [68]. Pus and infected peritoneal samples, for example, contain an average of 2*108 CFU/ml, a concentration 400 times higher Selleck Alectinib than the inocula used for standard conventional ASTs [68]. The β-LEAF assay is compatible with usage of high bacterial numbers

(i.e. ~108 CFU and higher), by virtue of which it may facilitate assessments at clinically relevant numbers based on infection sites. Some conventional AST methods, such as those relying on turbidometric detection of bacterial growth, may not Ulixertinib price be able to utilize higher bacterial numbers as the starting inoculum. Although PCR-based diagnostics have been employed to detect antibiotic resistance factors relatively rapidly [69–72], the presence of a gene does not necessarily reflect expression of the protein (e.g. enzyme), actually responsible for conferring enough resistance. For instance, Bacillus anthracis contains genes for lactamases bla1 and bla2, but usually resistance is not observed [73]. In the current study also, despite the different diagnostic methodologies for β-lactamase

enzyme production being consistent (nitrocefin disk test, zone edge test and the β-LEAF assay), the blaZ genotype did not match for some of the isolates (Table 2). In these isolates (e.g. #9, #15) no β-lactamase production was observed, although they contained the gene for β-lactamase (blaZ). Thus, investigating the protein resistance factor phenotypically can be of value. Rapid determination of functional β-lactamase and its correlation to antibiotic activity/usability by assaying for enzyme activity is a distinctive feature of the β-LEAF assay. Conclusions This study reports a fluorescence quenching-dequenching guided method for rapid β-lactamase detection and prediction of antibiotic activity in the context of β-lactamase. The initial results with standard ATCC bacterial strains and clinical isolates are encouraging, though further validation in a large number of isolates is required. The technology merits further rigorous and broader investigations with bacterial strains, antibiotics and direct biological samples to be a viable routine methodology. This requires the development of more sensitive probes and perhaps some novel engineering, which are currently being evaluated.

Acta Chir Belg 2004, 104:445–447. Selumetinib cost 35. Chalya PL, Mabula JB, Koy M, Kataraihya JB, Hyasinta Jaka H, Mshana SE, Mirambo M, Mchembe MD MD, Giiti G, Gilyoma JM: Typhoid intestinal perforations at a University teaching

hospital in Northwestern Tanzania: A surgical experience of 104 cases in a resource-limited setting. World J Emerg Surg 2012, 7:4.PubMedCrossRef 36. Thapa S, Satyal I, Malla K: Safe abortion service and post abortion care: understanding complications. N J Obstet Gynaecol 2007,2(1):44–49. 37. Saleem S, Fikree FF: Induced abortions in low socio-economic settlements of Karachi, Pakistan: rates and women’s perspectives. J Pak Med Assoc 2001,51(8):275–279.PubMed 38. Bhutta SZ, Aziz S, Korejo R: Surgical Complications following Unsafe Abortion. J Pak Med Assoc 2003, 53:286. 39. Ohanaka EC: Discharge against medical advice. Trop Doc 2002, 32:149–151. Competing interests The authors declare that they have no competing NVP-BGJ398 cell line interests. Authors contributions JBM conceived the study and participated in the literature search, writing of the manuscript and editing the article. PLC, MDM, GG, AK, AM, ABC, participated in Study design, data analysis, manuscript writing & editing. In addition PLC submitted the manuscript. JMG was

involved in study design, data analysis, coordination and supervision of manuscript writing & editing. All the authors read and approved the final manuscript.”
“Trauma is a major public health problem

worldwide. More than 5 million Dimethyl sulfoxide people die every year as a consequence of traumatic injuries. This disease does not distinguish between developed or underdeveloped countries; it is a major challenge to modern societies. In many instances, injuries occur due to their responsible actions such as drug abuse, drinking and driving, etc. Interpersonal violence, suicides, and motor vehicle crashes, just to name a few of the more prevalent mechanisms of injury, require aggressive prevention strategies. Social and economic inequalities leading to hunger, lack of access to healthcare, limited education, and violence are common in many underdeveloped countries. In those locations, the impact of injury is even greater and trauma care is sometimes neglected or inexistent. Trauma systems and adequate trauma care led by trauma / critical care / acute care surgeons are needed to fight this epidemic disease. The involvement of other health care groups: nursing, technicians, physical and occupational therapy, nutritional specialists, paramedics, emergency medical technicians, social workers, and medical specialists are paramount to provide comprehensive care to the injured patient.

tuberculosis-induced DNA fragmentation, as recommended by the manufacturer. Briefly, 1-3 days after infection, 48-well plates were centrifuged at 200 × g to sediment detached cells, the medium was discarded, and the cells were lysed. The lysate was subjected to antigen capture enzyme-linked immunosorbent assay Fulvestrant solubility dmso (ELISA) to measure free nucleosomes, and the optical density at 405 nm (OD405) was

read on a Victor2 plate reader (Wallac/Perkin Elmer, Waltham, MA). Triplicate wells were assayed for each condition. Staurosporine (Sigma) (1 μM, diluted in serum-free RPMI) was applied for 24 h as a positive control for DNA fragmentation. Caspase Inhibition The pan-caspase inhibitor, Q-VD-OPh (20 μM; Enzo Life Sciences AG, Lausen, Switzerland), was applied to

DCs 4 h prior to infection with H37Ra and replenished every 24 h throughout the duration of infection Caspase-Glo Assay Caspase 3/7 activity was measured using the luminescent Caspase-Glo assay system (Promega, Madison, WI). DCs were cultured in 96-well plates and the assays were carried out in a total volume of 200 μl. After equilibration to room temperature, Caspase-Glo reagent was added to each well and gently mixed using a plate shaker at 300 rpm for 30 s. The plate was incubated at room temperature for 30 minutes and luminescence was then Compound Library measured

using a Victor2 plate reader. Laser Scanning Confocal Microscopy Following infection, DCs were fixed for 10 min (H37Ra) or 24 h (H37Rv) in 2% paraformaldehyde (Sigma), applied to glass slides and left to air dry overnight. The cells were then stained with modified auramine O stain for acid-fast bacteria and DC nuclei were counterstained with 10 μg/ml of Hoechst 33358. The slides were analysed using a Zeiss LSM 510 laser confocal microscope equipped with an Argon (488 nm excitation line; 510 nm through emission detection) laser and a diode pulsed solid state laser (excitation 561 nm; emission 572 nm long pass filter) (Carl Zeiss MicroImaging GmbH, Oberkochen, Germany). Images were generated and viewed using LSM Image Browser (Carl Zeiss MicroImaging). Flow Cytometry Dendritic cell surface markers were analysed by flow cytometry on a CyAn ADP flow cytometer (Dako/Beckman Coulter). Dendritic cells were infected with live H37Ra, or streptomycin-killed H37Ra at MOI 1 for 24 or 48 h. As a positive control for maturation, uninfected DCs were treated with LPS (Sigma; 1 μg/ml) for 24 h prior to staining for flow cytometry. Cells were incubated with antibodies for 30 min and fixed with 2% paraformaldehyde for at least 1 h prior to flow cytometry.

It is intriguing that all of the organisms identified to date that have homologs of mglA also carry structural genes for the assembly of Tfp. Anaeromyxobacter dehalogens [52], Geobacter metallireducens [53], and members of the related genera Deinococcus and Thermus, have genes encoding Tfp [54]. Similarly, some genes for Tfp determinants are found in the genome of the filamentous glider Chloroflexus aurantiacus, an anoxygenic, thermophilic photosynthetic bacterium [55]. Not all organisms that have Tfp

have an mglA homolog, nor is it clear that all organisms encoding Tfp use these components for motility. buy Cobimetinib For example, Thermus thermophilus uses Tfp machinery for DNA check details transfer [56]. Future studies might reveal a novel pathway by which these unusual GTPases regulate Tfp components in organisms from diverse habitats and in diverse functions. Methods Strains and media Strains and plasmids are listed in Additional file 9: Table S1. M. xanthus strains

were grown routinely in vegetative CTPM (1% Casitone, 10 mM Tris, 1 mM potassium phosphate, 5 mM MgSO4, final pH 7.6) medium at 32°. Unless otherwise noted, solid medium contained 1.5% agar. E. coli strains were grown in LB medium [57] at 37°, and were used for plasmid constructions and DNA purifications. When appropriate, media were supplemented with kanamycin sulfate (Kan; 40 μg/ml). Construction Florfenicol of plasmids with mutations in mglA and recombination of mutant alleles of mglA into the M. xanthus chromosome in single copy We performed the site-directed mutagenesis of mglA using the PCR-overlap extension method [29]. To make each mutation,

pairs of overlapping oligonucleotides, shown in Additional file 9: Table S1, were synthesized. The first round of PCR was done using each of two mutagenic oligonucleotides and each of two (flanking) oligonucleotides complementary either to the 5′ or 3′ ends of the mglBA operon, to amplify overlapping portions of this operon from pPLH325. Gel-purified PCR products were cloned into plasmid vector pCR2.1 Blunt TOPO and recombinant plasmids, otherwise isogenic with their parental plasmid, pPLH325, were recovered from KanR transformants of either E. coli JM107 or Top10. The presence of each mutation was confirmed by the analysis of the entire mglBA coding sequence by RFLP analysis and/or sequencing. These plasmids were introduced into the M. xanthus genome by homologous recombination. Examination of mglA transcription M. xanthus strains were grown to a density of 5 × 108 cells/ml in CTPM medium at 32°C, and harvested by centrifugation. Total mRNA was harvested using Trizol reagent RNA extraction protocol (Invitrogen). Each sample was then treated with 6 units of DNaseI (Fermentas) for 45 min to remove any potential genomic DNA contaminants.

Biofilms were stained with 1% crystal violet, washed with deionised water and quantitated by adding 95% ethanol followed by measurement of the absorbance (OD 595 nm) as per Stepanovic et al. [33]. Strains with no change in O.D over the control were classified non-biofilm producers, weak- (up to a 2 fold change), moderate- (up to 4 fold change) or strong- (greater than 4 fold

change) as per Strepanovic et al. [33] All tests were carried out in triplicate and the results were averaged. P. aeruginosa strain PAO1 was included as a positive control. Biofilms in a capillary flow reactor were grown in glass capillary tubes of square cross sections under continuous flow conditions. The capillaries selleck inhibitor had a nominal inside dimension of 900 μm and a wall thickness of 170 ± 10 μm (Friedrich & Dimmock, Millville, N.J., USA). The flow cell apparatus consisted of a vented medium feed carboy (four litre capacity), a flow break, a filtered air entry, a peristaltic pump (Watson-Marlow), the capillary and flow cell holder,

an inoculation port, and a waste carboy. The components were connected by silicone rubber tubing and were sterilised by autoclaving. A culture of gfp-P. aeruginosa was grown in LB overnight at 37°C Z IETD FMK in a shaking incubator at 140 rpm. A 100 μl aliquot of this culture was used to inoculate 10 ml of sterile LB broth in a 250 ml conical flask to achieve good aeration and the culture was grown at 37°C with shaking at 200 rpm for 3 h. The tubing was clamped downstream of the inoculation port and the capillary flow system was inoculated with 300 μl of this fresh culture. The tubing was then clamped upstream of the glass tube and the system was allowed to stand without a flow for 19 h to allow the cells to attach to the glass capillary at 37°C. After initial attachment, the flow of medium (1/10 strength LB, to avoid blockage of the capillary due to excessive biomass production) was adjusted to

a flow rate of 20 ml h-1. Bacterial staining of mixed biofilms consisting of biofilm+ and biofilm- isolates, were stained with 300 μl of a 5 mg l-1 rhodamine B (Kodak) solution in water. The stain solution was injected into the capillary reactor through the Tenoxicam inoculation port and the cells allowed to stain for 5 min. Biofilms were subsequently observed by confocal scanning laser microscopy with excitation and emission wavelengths of 540 nm at 625 nm respectively for rhodamine B and 475 nm and 510 for GFP. Scanning Electron Microscopy (SEM) Prior to SEM, samples were chemically fixed as follows: A 10 μl aliquot of an overnight culture, grown in LB broth at 37°C, with shaking at 140 rpm was placed in a round glass coverslip (10 mm diameter, Chance Proper Ltd., UK) with a 10 μl of fixative (3% glutaraldehyde in 0.1% sodium cacodylate, pH 7.3). The coverslips were previously coated with polylysine (Sigma-Aldrich) to assist adherence of bacterial cells.

e. down regulates several host responses) in comparison to the ubiquitous serovars [39]. The lower cytotoxicity and lack of IL-6 responses support this assumption. In contrast to the role in IL-6 induction, none of the mutants differed significantly from the wild type strains in induction of oxidative responses. This result suggested that flagellin was not important for induction of the oxidative response. Results on the role

of flagella and chemotaxis genes in Salmonella host pathogen interaction have been contradictory (compare [12] and [8] with [11]), and we purposely looked for a sensitive assay to show subtle differences between strains. Co-infection assays have been shown to be more sensitive than assays where strains are tested individually [40]. Using buy Navitoclax this assay, we found that flagella significantly www.selleckchem.com/products/PD-0332991.html influenced the number of bacteria that could be isolated from the spleen 4–5 days post oral infection of mice with S. Dublin, but not with S. Typhimurium. Chemotaxis genes were found to be dispensable in this assay, as previously reported for S. Typhimurium [11]. Animal welfare regulations dictated us to scarify mice when they were severely affected by infection, and this prevented us from using one single end-point of infection. Potentially, this may have influenced the competitive indexes for S. Typhimurium, since this serovar propagated at different speed at systemic sites depending

on the presence of flagella genes (see below). However, all mice were killed within a 24 hours period, and we do not believe that this significantly influenced our results. Like cheA mutation, mutation of cheR confers a constitutively smooth swimming phenotype. We have not included this gene in our investigation, and we cannot rule out that it may have a different role in host pathogen interaction than cheA. We have performed preliminary testing of an S. Dublin cheR mutant and found that it corresponds to cheA with respect to phenotypes in cell assays and oral challenge of mice (unpublished), however, we do not have S. Typhimurium results to compare it

to. Flagella have been found to be important for the outcome of oral infection with S. Typhimurium in streptomycin treated mice, which is a model for studies of the entero-pahtogenesis of Salmonella[41]. In this model flagella Cyclin-dependent kinase 3 are essential for initiation of inflammation, creating an environment in which Salmonella prevails over the normal flora, and in this model, chemotaxis genes were also essential for the outcome of infection. Cattle are the natural host for S. Dublin, and in addition to differences caused by the choice of animal model, studies have shown that virulence factors may differ depending on the host [42]. This must be taken into account when concluding on the current results. The changes in virulence observed when flagella were removed were relatively modest.

Establishment of radio-resistant cell line The method for establishing radio-resistant cell line by fractionated irradiation has been described previously[13]. Briefly, the cell line was first grown to approximately 60% confluence in 25-cm2 culture flasks. Cells were irradiated with 10 Gy of X-ray irradiation, from a linear accelerator (6-MV X-ray), at a rate of 3 Gy/min. One cm thick of tissue-equivalent bolus was placed on top of the plate to ensure homogeneity. And then cells were returned to the incubator. When they reached approximately 60% confluence,

the cells were again irradiated with 10 Gy of X-ray. The fractionated irradiations were continued until the total concentration reached 80 Gy. The radio-resistant cell subline was find more then established. The parental cells were subjected to identical trypsinization, replating, and culture conditions,

but were not irradiated. For all assays on irradiated cells, there was at least a four-week interval between the last 10 Gy fractionated irradiation and the experiment. Assay for radiosensitivity Cell survival after X-ray irradiation was measured by clonogenic assay. Cells were plated in six-well culture plates, and were irradiated at different concentration ranging from 0 to 12 Gy. The appropriate plating density was aimed to produce 20–100 surviving colonies in each well. These cells were incubated at 37°C for 10–14 days (three wells in each radiation concentration). After fixation with acetic acid-methanol (1:4) and staining with diluted crystal violet (1:30), colonies consisting of selleck screening library 50 cells or more were counted under a light microscope. The triplicate colonies were averaged and divided by initial seeded cells to yield survival rate of clones for each concentration, and the surviving fraction was determined. All survival curves represent

at least three independent experiments. Detection of apoptotic cells Apoptosis was evaluated using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences Pharmingen, San Jose, CA, USA) followed by FACS analysis. Cells were treated with trypsin-EDTA in PBS at pH 7.5, washed with normal medium and cold PBS, and then resuspended in 1× binding buffer. Five μl of annexin V and ten μl of propidium iodide were added to the cells, vortexed, and incubated for 15 minutes in the dark. Finally, 400 μl of 1× binding buffer old was added, and samples were evaluated by flow cytometry. MTT cell viability assay Drug-induced cytotoxicity was evaluated by conventional MTT cell viability assay as previously reported [14, 15]. Briefly, 1 × 104/well EC109 or EC109/R cells were seeded in 96-well plates and cultured in DMEM media supplemented with 10% FBS for 8 h. They were exposed to various concentrations of cisplatin (3.33–63.3 μM), 5-fluorouracil (0.07–4.93 mM), doxorubicin (0.53–7.36 μM), paclitaxel (3.12–100 nM) or etoposide (1–16 μM) for 48 h in a CO2 incubator.