Because of the observational study design, the degree of inspiration during CT could not be controlled: www.selleckchem.com/products/chir-99021-ct99021-hcl.html Reference spontaneous patients were asked to hold their breath after inspiration (without checking for compliance) during CT. Reference ventilated and ALI patients were scanned during uninterrupted mechanical ventilation, which is current clinical practice in our institution. Calibration of the CT scanners was performed using air and the manufacturer’s standard phantom.Quantitative CT analysisThe lung parenchyma was segmented manually in CT images covering the entire lungs (Osiris software; University Hospital Geneva, Geneva, Switzerland) [25]. Window levels and widths appropriate for the lung parenchyma (-500/1,500 HU) or the mediastinum (50/250 HU) were used.

Major hilar vessels and bronchi, pneumothoraces, pleural fluids and gross motion artefacts were manually excluded. Only in aerated lung regions did we use a threshold (-350 HU)-based segmentation technique in an attempt to guide and standardize the manual exclusion of partial volume effects close to the thoracic wall, mediastinum, heart or diaphragm. To do so, window level and width were set to (-350/0 HU), and the segmentation line was drawn at the black-white interface [32-34]. Opacified lung regions were segmented manually using anatomical landmarks.The total lung volume (Vlung), the total lung mass (Mlung) and the masses of differently aerated lung compartments were calculated voxel-by-voxel using customized software as previously described [9,10,12,25,35].

Mlung and Vlung values were calculated on the basis of all lung voxels within the -1,000 to +100 HU range. The following HU ranges were used to separate differently aerated lung compartments: nonaerated, -100 to +100 HU; poorly aerated, -101 to -500 HU; normally aerated, -501 to -900 HU; and hyperaerated, -901 to -1,000 HU. The masses of differently aerated lung compartments were calculated as percentages of Mlung. Although it was calculated, we omitted between-group comparison of the hyperaerated compartment because two different CT scanners and image reconstruction protocols were used, and such comparison was not required for the present study [30].The validity of our analytical method was reviewed in 27 patients by placing a water-filled plastic bottle next to the thorax. We then selected an arbitrary region of interest (ROI) within this bottle in the CT image and compared the weight resulting from our voxel-by-voxel analysis method with that obtained by simply multiplying the volume of interest (ROI area �� slice thickness) by the volumetric mass density of water (approximately 997.77 kg/m3 at Brefeldin_A 22��C).Statistical analysisData are given as medians with interquartile ranges unless specified otherwise.

, India. The pharmaceutical dosage form used in this study was a Neucam-P (Lupin DAPT secretase clinical (maxter) Laboratories, Mumbai, India) and Lornicam plus 8 (Aristo Pharmaceutical Pvt. Ltd, Delhi, India) tablets containing 500 mg PCM and 8 mg LOX were purchased from the local drug market. Triple distilled water was generated in house. Chromatographic condition The isocratic mobile phase consisted of methanol-phosphate buffer (pH 7.0) in the ratio of (60:40 v/v), flowing through the column at a constant flow rate of 1.0 ml/ min. A Luna C18 column (5 ��m, 150mm �� 4.60mm) was used as the stationary phase. Although the PCM and LOX have different ��max viz 248 and 380, 290, 261 nm, respectively, but considering the chromatographic parameter, sensitivity and selectivity of method for two drugs, 260 nm was selected as the detection wavelength for UV-PDA detector.

Standard preparation Standard stock solution Standard stock solutions were prepared by dissolving separately 100 mg of each drug in 100 ml of diluent which was a mixture of methanol and phosphate buffer in the ratio of 60:40 (pH 7.0) to get concentration of 1000 ��g/ ml. Working standard solution Working standard solutions were prepared by taking dilutions ranging from 10-50, 8-40 ��g/ml for PCM and LOX, respectively. Sample preparation Twenty tablets of each brad Neucam-P and Lornicam plus 8 were weighed individually and ground to a fine powder. An accurately weighed powder sample equivalent to 8 mg of LOX and 500 mg PCM were transferred to 100 ml of volumetric flask. Drug was extracted with three 20 ml quantities of mixture of diluent.

The flask was sonicated for about 10 min to solubilize the drug and the volume was made up to the mark and filtered through Whatman filter paper No. 42, finally different concentrations of tablet sample were prepared by serial dilution technique. RESULTS AND DISCUSSION Chromatography The mobile phase was chosen after several trials with methanol, isopropyl alcohol, acetonitrile, water and buffer solutions in various proportions and at different pH values. A mobile phase consisting of methanol/phosphate buffer (60:40, v/v, pH 7.0) was selected to achieve maximum separation and sensitivity. Flow rates between 0.5 and 1.5/min were studied. A flow rate of 1.0 ml/min gave an optimal signal to noise ratio with a reasonable separation time. Using a RP C18 column, the retention times for PCM and LOX were observed to be 2.

06 and 4.38 AV-951 min, respectively. Total time of analysis was less than 5 min. The maximum absorption of PCM and LOX together as detected at 260 nm and this wavelength was chosen for the analysis [Figure 2]. Figure 2 Chromatograph resulting from (a) mobile phase (b) standard paracetamol (30 ��g) and lornoxicam (24 ��g) (c) standard paracetamol (50 ��g) and lornoxicam (40 ��g) with Rf min 2.06��0.013 and 4.38��0.07 min, respectively …

The implementation of metalloproteinases and their inhibitors as new biomarkers for the severity of sepsis and for mortality in critically ill patients may provide promising decision support Abiraterone for the intensivist to guide the allocation of hospital resources. Additional larger studies are needed, however, to determine the cellular origin and the relevance of these enzymes in sepsis.AbbreviationsIL: interleukin; MMP: matrix metalloproteinase; TIMP: tissue inhibitor of matrix metalloproteinase; TNF: tumor necrosis factor.Competing interestsMBr is an employee of Boehringer Ingelheim GmbH, Germany. The other authors declare that they have no competing interests.NotesSee related research by Lorente et al., http://ccforum.com/content/13/5/R158, and related letter by Lorente et al., http://ccforum.

com/content/14/1/402AcknowledgementsThe present work was supported by a grant of the Faculty of Medicine Mannheim, University of Heidelberg, Germany.
An increasing number of people in economically developed nations are receiving oral anticoagulants for the treatment and prophylaxis of thromboembolic diseases [1,2]. Among the most commonly used oral anticoagulants are the synthetic coumarin derivatives warfarin, acenocoumarol and phenprocoumon. All three drugs act by inhibiting the biosynthesis of the vitamin K-dependent clotting factors (factors II, VII, IX and X), which produces a functional deficit of these procoagulant proteins [2].

The main indications for vitamin K antagonists are: primary and secondary prevention of venous thromboembolism; prevention of systemic embolism (for example, stroke) in patients with mechanical heart valves or atrial fibrillation; and prophylaxis (as adjunctive therapy) for systemic embolism following myocardial infarction [3].While the antithrombotic benefits of oral anticoagulants are well established, these therapies increase the risk of hemorrhagic events, some of which may be severe or even life-threatening [4-7]. The risk of bleeding in patients receiving anticoagulants increases with surgery, trauma, over-anticoagulation or raised international normalized ratios (INRs) – although complications can still occur when the INR is within the therapeutic range [1,2,4,5,8-10].Because of the association between vitamin K antagonists and an increased risk of hemorrhagic events, patients undergoing emergency procedures and those with life-threatening/major bleeding or highly elevated INRs require urgent and immediate reversal of anticoagulant activity [1,5,11].

Recommended treatments for rapid reversal of oral anticoagulant therapy include fresh frozen plasma (FFP) and prothrombin complex concentrates (PCCs); in all cases these should be supplemented with oral or intravenous vitamin K [1,3,5,11-17]. PCCs, which contain three or four vitamin K-dependent clotting factors, offer a number of advantages AV-951 over FFP.

Figure 1Patient study recruitment diagram. ICU, intensive care unit.Table 1Patient characteristicsA total of 112 (27%) patients either refused to participate or selleck EPZ-5676 did not respond. These patients were significantly younger (42.4 years standard deviation (SD) 15.5 vs. 47.7 years SD 15.6, P = 0.003) and were more often transferred to local hospitals while still on MV, (49.1% vs. 26.6%, P = 0.001) than the patients that participated at four to six weeks (n = 255), but did not differ according to clinical variables.In the present study, the 43 patients who participated only at 12 months lack baseline data and were not included in the regression analyses, in the analyses of the course of symptoms or in the analyses of prevalence. The results from these patients (n = 43) were only used for comparisons with the responders (n = 194).

These patients were probably more seriously ill during the ICU stay because they had higher mean NEMS score (32.0, 95%CI = 30.4 to 33.7, vs. 29.6, 95% CI = 28.8 to 30.5; P = 0.04), were more often MV (97.7% vs. 84.7%, P = 0.02), had longer duration (days) of MV (16.2, 95% CI = 11.7 to 20.6, vs. 11.0, 95% CI = 9.3 to 12.7; P = 0.02) and were more often trauma patients (48.8% vs. 33.7%, P = 0.04) compared with the patients that participated at one month. No significant differences were found in age, gender, SAPS, LOS ICU, head injury/diseases or the proportion of patients that were transferred to other hospitals. The 43 patients did not differ significantly from the 194 patients at the one-year measurements of IES-total (21.9 vs. 22.5), HADS-Anxiety (6.

8 vs. 5.8) or HADS-Depression (5.4 vs. 4.4) scores.The level of psychological distressThe mean score for IES-total one-year after ICU discharge (Table (Table2)2) was not significantly different between genders, but woman had higher scores than men (25.4 for women, 95% CI = 20.8 to 30.0, vs. 20.8 for men, 95% CI = 17.7 to 23.9; P = 0.086). Twenty-seven percent of the patients had scores at PTSD level at one year (IES-total �� 35; Table Table2).2). No significant differences in psychological distress symptoms were seen between medical, surgical and trauma patients at one year, except that slightly more surgical patients had a HADS-Depression score of 11 or more compared with medical and trauma patients.

Table 2Psychological distress measurements at one yearDuring the first year following ICU discharge no differences in the IES-total, HADS-Anxiety and HADS-Depression mean scores across the three time points were found (Friedman, P = 0.388, P = 0.076, P = 0.446, respectively). Neither did we find any difference in the Batimastat percentage of patients with symptoms above the lowest cut-off value, IES-total of 20 or more, between baseline (46%) and 12 months (51%; n = 170; Figure Figure2).2).

The purpose of this study Oligomycin A IC50 was to develop simple, fast, economical and validated analytical methods to quantify repaglinide in tablets using HPLC and UV spectrophotometry. Validation of the developed methods was done as per International Conference on Harmonization (ICH) guidelines.[11] The results obtained by these methods were statistically compared using analysis of variance. In addition the reliability and feasibility of these methods were evaluated, focusing on rountine quality control analysis. MATERIALS AND METHODS Materials Repaglinide, reference standard was obtained as a generous gift sample from USV Lab. Pvt. Ltd., Mumbai, India. Eurepa tablets labelled to contain repaglinide 2 mg, manufactured by M/S Torrent Pharmaceutical Ltd., Baddi (H.P.), India, were purchased from local market.

All the chemicals used were of AR and HPLC grade, obtained from E. Merck, India. Instrumentation and optimization conditions UV spectrophotometric analyses were carried out on a Shimadzu 1700 Double beam UV-Vis spectrophotometer, with 1.0-cm quartz cells. The wavelength of 241 nm was selected for the quantitation of repaglinide and the measurements were obtained against methanol as a blank. The HPLC analysis were carried out on Agilent 1120 Compact LC system composed of binary pump, manual injector, UV detector and Ezchrome EliteCompact software. The column used was Agilent TC-C18 (250 mm �� 4.6 mm i.d., 5 ��m particle size) and the mobile phase consisted of methanol and water (80:20 v/v, pH adjusted to 3.5 with orthophosphoric acid) at a flow rate of 1.0 ml/min.

Detection was performed on at 241 nm. Preparation of standard solution The standard stock solution of repaglinide 1000 ��g/ ml was prepared in methanol. For spectrophotometric method, further dilutions of aliquots of standard stock solution were carried out with methanol to reach the concentration range 5-30 ��g/ml for repaglinide. The absorbance of series of solutions was measured at 241 nm and found to be proportional to the corresponding concentrations of repaglinide. For HPLC method, the standard solutions were prepared by dilution of aliquots of the standard stock solution with mobile phase to reach the linearity range of 5-50 ��g/ml of repaglinide. Twenty microlitre of the each standard solution was injected to HPLC system. The peak areas were plotted against the corresponding concentrations to obtain the calibration graph.

Preparation of sample solution To determine the content of repaglinide in conventional tablets (label claim: 2 mg repaglinide per tablet), 20 tablets were weighed, their mean weight determined and finely powdered. A portion of tablet powder equivalent to 10 mg of repaglinide was accurately Dacomitinib weighed and dissolved in 30 ml methanol in 100 ml volumetric flask. The contents of the flask were sonicated for 15 min to dissolve repaglinide, volume was made up to the mark with same diluent and the resulting mixture was filtered.

In the present study, we investi gated the relationship between NF ��B and STAT3 in terms of gastric cancer metastasis. To the best of our knowledge, this is the first study to show the associ ation between NF ��B and STAT3 in gastric www.selleckchem.com/products/U0126.html cancer. In the present study, constitutive activation of NF ��B and STAT3 was found in 16% and 24% of 255 gastric cancer specimens, respectively, and they showed a positive correlation. In addition, our in vitro experiments showed that NF ��B inhibition reduced the protein expres sion of total STAT3 and pSTAT3, which was possibly caused by the suppression of STAT3 at the transcriptional or translational level. Since we wondered whether there is a reciprocal regulatory loop between NF ��B and STAT3, we further analyzed the effect of STAT3 silencing on the NF ��B activation.

However, we found that STAT3 did not affect either NF ��B expression or activation. Thus, these results suggest that STAT3 is a downstream mol ecule of NF ��B in NF ��B pathway. Our observations contrast with a report by Yang et al. which showed that STAT3 and RelA can heterodimerize to transcriptionally regulate NF ��B dependent genes. Although Wani et al. reported that NF ��B activa tion induced STAT3 activation mediated by IL 6, the present study did not show whether IL 6 is reduced in the SNU 638 cells overexpressing I��BM, which may account for the reduced STAT3 levels. Thus, fur ther investigations are needed to obtain a better under standing of the mechanism involved in NF ��B induced STAT3 activation. EMT confers acquisition of cell migration and invasion as well as molecular alterations in cancer cells.

Al though the existence of EMT has not been shown in all types of cancers, previous studies have demonstrated that EMT plays a key role in the malignant progression of gastric cancer by using gastric cancer cell lines, ortho topic xenograft tumors and surgical gastric cancer speci mens. In the present study, we showed that I��BM overexpression decreased the migration and in vasion of gastric cancer cells. Moreover, I��BM overepx ression increased E cadherin expression and decreased Snail expression, which indicates the change toward the mesenchymal phenotype. Thus, these results indicate that NF ��B might contribute to malignant progression through promotion of EMT. Regarding the role of STAT3 in gastric cancer cells, Okamoto et al.

found that STAT3 activation induced cancer cell motility through the Janus kinase pathway, whereas it enhanced survival of MET activated gastric cancer cells. Thus, they concluded that STAT3 plays differential roles depending on the upstream regulator of STAT3 activation in gastric cancer cells. In the present study, STAT3 silencing decreased the migration and inva sion in SNU 638 gastric cancer cells with high Drug_discovery NF ��B activity.

The quantity of expressed protein was normalized to GAPDH. Proliferation assay Cell proliferation assays were performed using Cell CountingKit 8. Cells were plated in 96 well plates at 3. 5��103 cells Erlotinib side effects per well and cultured in growth medium with 2% FBS. At the indicated time points, the cell numbers in triplicate wells were measured as the absorbance at 450 nm from WST 8 3 5 2H tetrazolium, monosodium salt Boyden chamber migration Cell migration assays were performed using Millicell cell culture inserts. HBSMCs, which had been treated with siRNA for 48 h, were serum starved overnight. PDGF BB and 10% FBS were prepared in SmGM and added to the bottom cham bers. HBSMCs in serum free SmGM were added to the upper chambers. After 5 h of incubation at 37 C, cells on both sides of the membrane were fixed and stained with 0.

1% crystal violet. Cells on the upper side of the membrane were removed with a cotton swab. The average number of cells per field was determined by counting the number of cells in four high power fields from the lower side of the membrane. Gel contraction assay The contractility of the cultured HBSMCs was examined using a gel contraction assay. For each 6 well plate, collagen solution was prepared by mixing 450 ul of ice cold type I collagen with 53 ul 10�� PBS, pH was adjusted to 7. 4 with 0. 1 M NaOH. HBSMCs pretreated with siRNA for 48 h were seeded at a density of 3��105 cells ml, 1. 5 ml of gel suspension was poured into a 6 well culture pate. The gels were cultured in 2 ml of 5% FBS SmGM overnight added with PDGF or PBS and then started the contrac tion assay.

Gel surface images were captured with a digi tal camera 24 h later. Contraction of the gel was then evaluated by measuring its surface area with Image Pro Plus 6. 0. Data were expressed as percentage of the original gel size. Proteomic analysis Proteomic analysis was performed, as previously described. Briefly, HBSMCs transfected with NEGi or NOGOi 2 from three 60 mm cell culture dishes were, respectively, pooled as one sample. Total proteins of the cell samples were homogenized and treated with 2 D Clean Up Kit, following the manufacturers protocol. Protein from each sample was loaded into DryStripTM and iso electric focusing was performed on MultiphorTMII at 18 C. Two 15 min equilibration steps were carried out using equilibration tubes.

After equili bration, the strips were transferred onto 15% polyacryla mide gels for second dimensional SDS PAGE. The 2ndD gels were silver stained and digitized using an ima ging system Anacetrapib ChemiImagerTM 5500. Image analysis was conducted using the ImageMasterTM 5. 0. Only significantly different spots were selected for analysis by mass spectrometry. Target pro teins were excised and digested. Peptides were then extracted, dried and subjected to MALDI TOF MS analy sis.

To address this question, we repeated the starva tion experiment but re fed the myotubes in either the differentiation medium, serum free AMEM, or starvation medium supplemented with leucine, dialyzed FBS or horse therefore serum. Only media that contained serum promoted the degradation of PDCD4. Association of PDCD4 with eIF4A in L6 myotubes is sensitive to medium composition PDCD4 inhibits mRNA translation initiation at least in part by its binding to eIF4A and eIF4G. The amount of PDCD4 found in eIF4A immunoprecipitate was increased by starvation but fell gradually during refeeding, especially at 3 h, at which time the values were not different from those observed in fed cells. In another ex periment, we carried out the reciprocal immunoprecipita tion.

The amount of eIF4A in PDCD4 immunoprecipitate was unchanged by treatments, however, because starvation the interactions. In all cases, the effect of refeeding on the interactions of PDCD4 with eIF4A and 4G was sensitive to rapamycin. PDCD4 depletion in myotubes had modest effect on protein synthesis To examine the significance of PDCD4 in regulating myotube mixed protein synthesis, we used RNAi to de plete this protein and then measured incorp oration of phenylalanine into myotube proteins. In fed cells, incorporation of phenylalanine into mixed proteins in cells deprived of PDCD4 was not different from the value in those treated with scrambled oligonu cleotides. In cells deprived of serum but supplied with amino acids, phenylalanine incorporation into proteins in cells treated with PDCD4 siRNA 1 was 86% of values in those treated with scrambled siRNA, the values in those treated with PDCD4 siRNA 2 was 67% of those treated with scrambled siRNA.

In another experiment, PDCD4 deprived cells were incubated in medium lacking both serum and amino acids. Incorporation of phenylalan ine into myotube total mixed proteins in cells treated with the two PDCD4 siRNA oligonucleotides was 72 80% of the values in cells treated with scrambled siRNA oligonucleotides. Finally we examined the effect of PDCD4 on the regulation of myofibrillar proteins. Depletion of PDCD4 led to a 30% reduction in phenylalanine incorporation into myofibrillar proteins. The finding of reduced protein synthesis in cells de prived of PDCD4 was surprising given the inhibitory role of this protein on mRNA translation and our previous finding in myoblast. Thus we carried out two additional control experiments. First, we repeated the myoblast experiments and showed that as before, in starved cells, PDCD4 depletion increased protein synthesis by 43%. Finally, we used siRNA oligonucleotides purchased from another company to silence PDCD4 in myo tubes. Protein synthesis in myotubes Dacomitinib deprived of PDCD4 was reduced by 21%.

Each data point itself is the bulk average of a large number of cells, and so it is assumed that the sample average from this large collection of cells is normally distributed with mean equal to the popula tion average, but that the standard deviation can vary with time. Individual samples are assumed to be indepen Parameter values were estimated by minimizing sellekchem the a cost function based on the goodness of fit between model and data. Two objective functions were used, one which computed the normalized sum of squares error, between the model simulations at parameter set, y, and observed data points yobs, where i indexes the n time points at which data was collected. A second objective function used the chi square test statistic com puted from Fishers method, an adaptation of the moment matching algorithm proposed in.

The simulated concentrations of NF B and IKK were nor malized to their respective concentrations at 20 min and 5 min to allow direct comparison with experimental data. Optimization was performed using the fmincon constrained minimization algorithm from the Matlab Optimization Toolbox. Lower and upper bounds for the parameter values were taken from the available literature, as specified in Additional file 1. The normalized first order sensitivity coefficients of the system, dent across experiment replicates and identically distrib uted with regard to their respective time points. This is justified since all samples are collected from independent cell populations.

Under these assumptions, a two sided one sample t test can be used to compare the population mean from the model simulations corresponding to a specific set of parameters, to the sample mean from ni experimental samples collected at time ti. The null hypothesis that the two are consistent is rejected at a significance level a if the p value corresponding to the ith t statistic is pi a. Fishers method combines the information from the individual test results to test the shared null hypothesis that all the ni experimental samples come from cell populations whose time evolution of the population average is given by the kinetic model. The test statistic for Fishers method is computed by combining each independent test as follows, n where yi is a system output andj is the jth rate para meter, were solved using the CVODES forward sensitiv ity solver from the SUNDIALS 2. 4.

0 software suite. Sensitivity scores were also assigned based on the time averaged integral of the normalized sensitivity magnitudes, The biological literature represents the repository of bio logical knowledge. The ever increasing scientific litera ture now available electronically and the exponential growth of large scale molecular data AV-951 have prompted active research in biological text mining and information extraction to facilitate literature based curation of mole cular databases and biomedical ontologies.

All of the reac tions were performed in 50 uL reaction volumes in trip licate. Standard curves were generated for gC1qR and B actin. The B actin gene was used as an internal selleck chemicals llc control in all of the PCR e periments. Western blot analysis After various treatments, cells were harvested, pelleted by short centrifugation and suspended in lysis buffer sup plemented with protease inhibitors for 30 min on ice. The supernatants were collected by centrifugation at 13,000 g at 4 C for 15 min. An equal amount of pro tein was separated by SDS PAGE on a 10 15% polyacryl amide gel and transferred to a PVDF membrane. The transferred membranes were blocked for 1 h in 5% non fat milk in PBST, in cubated with appropriate primary antibodies followed by horseradish pero idase conjugated secondary antibodies.

The protein bands were visualised using the enhanced chemiluminescence Western Detection System. Cell viability analysis The water soluble tetrazolium salt assay was performed to as sess C33a and SiHa cell viability. The WST 1 assay is a colorimetric method in which the dye intensity is propor tional to the number of viable cells. Cells were seeded into 96 well microtitre plates at a concentration of 5 103 cells well. After 12 h of incubation, cells were treated with for 48 h. After incubation, the cells were washed with PBS, WST 1 cell proliferation reagent was added, and the sam ples were incubated for 4 h. Sample absorbance was analysed with a bichromatic ELISA reader at 450 nm. All of the e periments were performed in triplicate with dif ferent C33a and SiHa cell passages.

Cell migration analysis C33a and SiHa cell migration was measured using 24 mm diameter chambers with 8 um pore filters. Cells were collected and resuspended in serum free media, and a 0. 2 mL cell suspension was added to the upper cham bers. Treatment media was added to the lower chambers. The chambers were incubated for 48 h at 37 C in a humidified atmosphere of 5% CO2 95% air. Ne t, the filters were fi ed in 95% ethanol and stained with H E. The upper filter surfaces were scraped twice with cotton swabs to remove non migrated cells. E peri ments were repeated in triplicate with different passages of the C33a and SiHa cells, and the migrated cells were counted microscopically in five different fields per filter. Apoptotic cell detection C33a and SiHa cell apoptosis was detected using the Anne in V FITC propidium iodide staining kit via flow cytometry.

After different treatments at the indi cated times, C33a AV-951 and SiHa cell were washed and resuspended in binding buffer before being trans ferred to a 5 mL tube. The cells were incubated in the dark with 5 uL each of Anne in V FITC and propidium iodide for 15 min. Binding buffer was then added to each tube, and the samples were analysed using a Beckman Coulter Epics L flow cytometer.