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PubMed2006

Optical absorption coefficient, time of thermal relaxation, time of surface threshold, and time of heat incubation for PMMA samples at the CO2 laser-beam wavelength of 10.6 microm.

OBJECTIVE: This paper discusses in detail the mathematical identification of the optical absorption alpha (cm(1)) of Beer's law, a crucial parameter to study the development of laser beam craters into dry poly(methyl methacrylate) (PMMA) samples exposed to steady CO(2) laser beams emitting radiation at lambda = 10.6 microm in continuous- wave (CW) mode. Three additional time-dependent coefficients have been determined as well. In clinical applications, these results are important in order to precisely quantify and forecast the ablation capabilities of the CO(2) laser beam, to optimize its usage in the operating room, and to address the safety issues related to surgical interventions on human tissue. BACKGROUND DATA: Currently, the data available in the literature do not allow the identification of the numerical value of alpha (cm(1)) for PMMA at lambda = 10.6 microm with enough, and therefore satisfactory, accuracy. Additionally, the correct identification of the optical absorption of PMMA would allow the isolation, with better accuracy, of other key time-dependent coefficients, such as relaxation time, surface threshold time, and heat incubation time, which are all described in the literature in a qualitative rather than quantitative fashion. Correct bone cement preparation depends on the value of alpha (cm(1)) of the PMMA in order to avoid unwanted complications in patients during cement removal via laser techniques. METHODS: The laser in use was configured in different combinations with the following parameters: transverse electromagnetic modes (TEMnm), output power (I0), exposure times (te), and focal lengths (fk). Several PMMA blocks (1 cm x 4 cm x 4 cm) were exposed to CW radiation of three commercially available CO(2) medical laser devices showing a TEM11 mode. Each block was exposed to the beam on a horizontal and well-polished surface of each sample. Four focal lengths (2.5", 5", 7.5", and 15.75" [400 mm]) were used to focus the beam on the well-polished and dry surface of the PMMA samples. The resulting dimensions of the craters were measured after each exposure, which has been kept at a 10-Watt CW beam. Exposure time ranged from 0.5 to 2 sec. RESULTS: The value of alpha = 502 (cm(1)) for PMMAat 10.6 microm was identified, matching other results reported in the literature for similar compact media in the absence of water content, such as PMMA. The time of thermal relaxation was 9.358 x 10(4) sec, the time of surface threshold was 9.365 x 10(4) sec, and the time of heat incubation was 3.6 x 10(7) sec (all three for PMMAat 10.6 microm for any exposure). Using the calculated value of alpha, one of the practical clinical recommendations would be, for instance, to reduce or to abolish the utilization of colorant dopants in the preparation of the bone cement mixture and therefore reduce the danger of bone damage possible during the removal of bone cement via laser techniques. Other examples refer to other clinical bone and dental treatments.

Bone Cements↗
PubMed2006

Distant metastases in head and neck carcinoma: identification of prognostic groups with MR imaging.

PURPOSE: To evaluate retrospectively the prognostic significance of lymph node parameters assessed on pretreatment magnetic resonance (MR) images for development of distant metastases in patients with head and neck squamous cell carcinomas. MATERIALS AND METHODS: Pretreatment MR images of 311 patients were retrospectively reviewed for the presence of lymph nodes at specific neck node levels as well as the size and the presence of a number of lymph node characteristics including extranodal spread, central necrosis and number and volume of ipsi- and contralateral nodes. Of these patients, 174 (56%) had MRI-positive nodes (defined as nodes with minimum axial diameter >8 and >4 mm for paratracheal level and retropharyngeal nodes). RESULTS: The 2-year distant-metastasis free survival rate (DMFSR) for patients without MRI-positive nodes was 94% compared to 75% for those patients with MRI-positive nodes. In patients with MRI-positive nodes, results of multivariate analysis with the Cox regression model yielded statistical significance for presence of extranodal spread (ENS), detected on MRI, as the only independent prognostic factor associated with the 2-year DMFSR (p=0.002). Based on the analysis, three risk groups regarding the DMFSR could be identified. Low-risk group (DMFSR:94%) consisted of patients without MRI-positive nodes. Intermediate-risk group (DMFSR:81%) consisted of patients with MRI-positive nodes without ENS. High-risk group (DMFSR:59%) consisted of patients with MRI-positive nodes and ENS as shown on MRI (p<0.0001). Statistical separation for different tumor locations showed MRI-determined ENS (larynx: p=0.05; oropharynx: p=0.04; oral cavity: p<0.001), lowjugular/posterior triangle nodes (oropharynx: p=0.02), paratracheal nodes (larynx: p=0.03), and contralateral node volume >5 cm3 (larynx: p=0.03; oral cavity: p=0.02) to be significant predictors with regard to DMFSR. CONCLUSION: Especially patients with on MRI demonstrating extranodal spread and with suspicious nodes at lowjugular/posterior triangle (oropharyngeal cancer) or paratracheal level (laryngeal cancer), or with contralateral enlarged nodes (laryngeal and oral cavity cancer) are at high risk for developing distant metastases and this subset of patients might benefit from supplementary imaging screening (CT-chest, PET-scan).

Carcinoma, Squamous Cell↗
PubMed1986

DNA-flow cytometry of squamous cell carcinomas from the human uterine cervix: the identification of prognostically different subgroups.

The relevance of DNA-index, alone and in combination with other parameters, was assessed for patients with squamous cell carcinomas of the uterine cervix by using the method of flow cytometry (FCM). Tumours with a diploid or a tetraploid DNA-content appeared to be associated with a less favourable prognosis than was observed in the group with non-diploid/non-tetraploid tumours. DNA-index in combination with either the age of patients or the histological grade of tumours, enabled the separation of prognostically different subgroups. Particularly, the combination of an age under 51 years with a DNA-index of 1 or 2 appeared to define a group with a low survival rate. Furthermore, within the group of well and moderately differentiated tumours, it was possible to identify prognostically different subgroups by combining this parameter with DNA-index, i.e. a non-diploid/non-tetraploid DNA-content of these tumours was associated with a more favourable prognosis. From this study it is concluded that FCM clearly contributes to the recognition of subgroups of patients with different prognosis.

Adult↗
PubMed2002

Parameters for reliable results in genetic association studies in common disease.

It is increasingly apparent that the identification of true genetic associations in common multifactorial disease will require studies comprising thousands rather than the hundreds of individuals employed to date. Using 2,873 families, we were unable to confirm a recently published association of the interleukin 12B gene in 422 type I diabetic families. These results emphasize the need for large datasets, small P values and independent replication if results are to be reliable.

3' Untranslated Regions↗
PubMed2006

Identification of critical process variables affecting particle size following precipitation using a supercritical fluid.

The critical processing parameters affecting average particle size, particle size distribution, yield, and level of residual carrier solvent using the supercritical anti-solvent method (SAS) were identified. Carbon dioxide was used as the supercritical fluid. Methylprednisolone acetate was used as the model solute in tetrahydrofuran. Parameters examined included pressure of the supercritical fluid, agitation rate, feed solution flow rate, impeller diameter, and nozzle design. Pressure was identified as the most important process parameter affecting average particle size, either through the effect of pressure on dispersion of the feed solution into the precipitation vessel or through the effect of pressure on solubility of drug in the CO2/organic solvent mixture. Agitation rate, impeller diameter, feed solution flow rate, and nozzle design had significant effects on particle size, which suggests that dispersion of the feed solution is important. Crimped HPLC tubing was the most effective method of introducing feed solution into the precipitation vessel, largely because it resulted in the least amount of clogging during the precipitation. Yields of 82% or greater were consistently produced and were not affected by the processing variables. Similarly, the level of residual solvent was independent of the processing variables and was present at 0.0002% wt/wt THF or less.

Carbon Dioxide↗
PubMed2004

[Identification of colorectal cancer using proteomic patterns in serum].

BACKGROUND & OBJECTIVE: At present there is no serological parameter with high sensitivity and specificity to diagnose colorectal cancer. This study aimed at establishing a serum protein fingerprinting technique coupled with a pattern-matching algorithm to distinguish colorectal cancer from benign colorectal diseases and healthy people. METHODS: Preliminary group and test group were both random selected serum samples, which each comprises 73 cases of colorectal cancer, 31 healthy people, and 16 cases of benign colorectal diseases. The sera of the test group was combined with the surface of the IMAC3 proteinchip. Then reading data of surface enhanced laser desorption/ionization- time of flight-mass spectrometry (SELDI-TOF-MS) was analyzed by Biomarker Wizard software and Biomarker pattern software to get a classification rule of tree, which is standard configuration that can distinguish the sera of colorectal cancer patients from the sera of benign colorectal disease patients and healthy people, and the standard was approved test validity by double-blindly in the test group. RESULTS: At the key M/Z values of 4 467 Da, 8 131 Da, 8 939 Da, 9 192 Da, 9 134 Da, 8 221 Da, 5 928 Da, 8 324 Da, 11 732 Da, protein contents of the three classes in preliminary group are obviously different by the software analysis. And the classification rule of tree was discovered, and corresponding correct ratio was 98.33% (118/120), corresponding sensitivity was 97.26% (71/73) and corresponding specificity was 100% (47/47); after double-blind examining the test group with that rule, the corresponding correct ratio was 96.77% (116/120),the corresponding sensitivity was 95.89% (70/73) and the corresponding specificity was 97.87%(46/47). CONCLUSION: Via comparative proteomics research in the sera of colorectal cancer patients and benign colorectal disease patients and healthy people, the group of protein was obviously different in pathological process in discovering colorectal cancer.

Algorithms↗
PubMed2004

Has the transition to agriculture reshaped the demographic structure of prehistoric populations? New evidence from the Levant.

This paper presents the demographic changes that followed the transition from a hunting-gathering way of life (Natufian) to an agricultural, food-producing economy (Neolithic) in the southern Levant. The study is based on 217 Natufian (10,500-8,300 BC) skeletons and 262 Neolithic (8,300-5,500 BC) skeletons. Age and sex identification were carried out, and life tables were constructed. A five-parameter competing hazard model developed by Siler ([1979] Ecology 60:750-757) was used to smooth life-table data. No indication of increased mortality with the advent of agriculture was noted. On the contrary, both life expectancy at birth (24.6 vs. 25.5 years) and adults' mean age at death (31.2 vs. 32.1 years) increased slightly from the Natufian to the Neolithic period (assuming stationary populations). Yet the transition to agriculture affected males and females differently: mean age at death in the Natufian was higher for adult females compared to adult males, while in the Neolithic, it was the reverse. One interpretation given to the distribution of female ages at death is that with the onset of the Neolithic period, maternal mortality increased as a result of a concomitant increase in fertility. If the adoption of agriculture in the Levant increased the rate of population growth at the beginning of the Neolithic, expectation of life may have increased dramatically.

Adolescent↗
PubMedDate not supplied

Single-cell detection by gradient echo 9.4 T MRI: a parametric study.

Recent studies have shown that cell migration can be monitored in vivo by magnetic resonance imaging after intracellular contrast agent incorporation. This is due to the dephasing effect on proton magnetization of the local magnetic field created by a labelled cell. Anionic iron oxide nanoparticles (AMNP) are among the most efficient and non-toxic contrast agents to be spontaneously taken up by a wide variety of cells. Here we measured the iron load and magnetization of HeLa tumour cells labelled with AMNP, as a function of the external magnetic field. High-resolution gradient echo 9.4 T MRI detected individual labelled cells, whereas spin echo sequences were poorly sensitive. We then conducted a systematic study in order to determine the gradient echo sequence parameters (echo time, cell magnetization and resolution) most suitable for in vivo identification of single cells.

Cell Survival↗
PubMed1976

Optimal temperatures for inner-ear performance in gekkonoid lizards.

Temperature effects on the cochlear alternating potentials are described in lizards of the Eublepharidae (4 spp. and subspp.), Diplodactylinae (2 spp.), Gekkoninae (7 spp. and subspp.) and Sphaerodactylidae (1 sp.). Pure tones (50-15,000 Hz) were applied to the anaesthetized gecko's external ear. Sound intensity needed for a standard response was plotted (db versus frequency). A total of 249 such sensitivity functions were obtained from 50 animals, at temperatures of 15-40 degrees C. For each species, an optimal sensitivity function is definable by its extreme sensitivity and relative smoothness. This is obtained throughout a specific optimal temperature range. At lower temperatures, the function is less sensitive and its best sensitivity is at a lower frequency. The specific optimal cochlear temperature range resembles the specific ecological temperature preferendum where known. Both are broader in gekkonoid than in iguanid lizards. The temperature effects on the sensitivity function occur even with aerial or mechanical stimulation at the oval window after middle ear extirpation. Hence they arise in the inner ear. Their magnitude (db/ degrees C) is half that known from auditory nerve potentials. When accurate auditory data are desired, the specific optimal temperature and its possible seasonal variation require consideration. This increases the need for proper identification of subjects by name and origin, and for monitoring experimental thermal parameters, including pre-experimental acclimation.

Acoustic Stimulation↗
PubMed1995

Single-base sequencing and similarity comparisons.

A "single-base sequence" is a DNA sequence in which the identities and locations of bases of only one type have been determined. We present experimental procedures for single-base sequencing and describe the effective use of existing software (FASTA) in similarity comparisons of single-base sequences. We determined the theoretical and experimental minimum sequence lengths required for identification of a sequence within a large dataset and optimized the FASTA parameters for use in single-base similarity comparisons. Single-base sequences have been used to identify cDNAs occurring in a database. Single-base sequencing could be used to reduce the redundancy of "shot-gun sequencing."

Databases, Factual↗
PubMed1993

Crystal and molecular structure of d(CGTAGATCTACG) at 2.25 A resolution.

Single crystal X-ray diffraction methods have been used to determine the structure of the synthetic DNA dodecamer d(CGTAGATCTACG). The dodecamer presents a new crystal form of B-DNA in the monoclinic system, space group C2 with a = 64.83 A, b = 35.36 A, c = 25.35 A and beta = 92.24 degrees. Structure solution was by molecular replacement and the refinement used a combination of rigid body treatment, molecular dynamics simulated annealing and restrained least-squares methods. The refinement has been concluded with an R-factor of 13.8% for 2120 reflections (78% of what is theoretically available) with F > or = 2 sigma (F) in the resolution range 7.0 to 2.25 A. The asymmetric unit comprises a B-form duplex (24 nucleotides, molecular weight 7.2 kDa), 136.5 water molecules and a single magnesium ion at 50% occupancy. The DNA model has root-mean-square derivations from standard bond lengths of 0.011 A. The central part of the structure is d(GATC), a tetrad sequence involved in methylation and mismatch repair. A comparison with other structures containing such a tetrad indicates polymorphism at the tetrad level. In our structure a narrow minor groove with a pronounced hydration pattern is observed. Such features may be of importance in the recognition of the sequence by specific enzymes. A noteworthy feature of the hydration is the identification of several pentagonal arrangements of hydrogen bonding groups. Details of conformational parameters, base stacking patterns, intermolecular interactions and hydration are presented and comparisons with related structures are given.

Base Sequence↗
PubMed1988

Use of commercially available monoclonal antibodies for immunoenzyme double staining.

An immunoenzyme double-staining method for the simultaneous detection of two cellular epitopes, using commercially available mouse monoclonal antibodies, is described. The method employs a combination of the suppression of endogenous biotin and two successive indirect techniques with a blocking step in between. The first indirect method involves an unlabelled monoclonal antibody followed by an alkaline phosphatase-conjugated goat anti-mouse immunoglobulin. After a blocking step with normal mouse serum, the second indirect method is applied using a biotinylated monoclonal antibody followed by the visualization of this antibody by avidin-biotinylated peroxidase complex (ABC) or rabbit anti-biotin and peroxidase-conjugated swine anti-rabbit immunoglobulin in successive steps. Using these methods in combination with the introduction of dioctyl sodium sulphosuccinate and tetramethylbenzidine as chromogens for peroxidase activity, two cellular epitopes could be distinguished clearly in tissue sections by the green- and violet-stained peroxidase and alkaline phosphatase activities, respectively. The expression of two epitopes on the same cellular constituent is outlined by the coappearance of both enzyme activities as a bluish-purple colour. This method allows for the simultaneous identification, localization and enumeration of two cellular epitopes. These can serve as parameters for a number of pathological processes.

Animals↗
PubMed1998

Pre-treatment energy status of primary rat tumours as the best predictor of response to 5-fluorouracil chemotherapy: a magnetic resonance spectroscopy study in vivo.

PURPOSE: Fluorine-19 magnetic resonance spectroscopy (19F-M RS) studies of the pharmacokinetics of the anticancer drug 5-fluorouracil (FU) in patients at several clinical centres have shown that increased tumour retention of FU is associated with patient response. The mechanism of this increased tumour retention (FU trapping) is unknown. We used a pre-clinical model to investigate whether other MRS-measurable parameters would correlate with the response to FU treatment and, thus, help elucidate the mechanism(s) involved in FU trapping. METHODS: MRS spectra were obtained using a double-tuned (31P/19F) surface coil from 29 N-methyl-N-nitrosourea-induced primary rat tumours. 31P-MRS spectra were acquired immediately prior to and at 2.5 h post-treatment with a bolus i.p. injection of FU (100 mg/ kg); 19F-MRS spectra were acquired during the intervening 2.5-h period for measurement of the tumour uptake and retention of FU and of its metabolism to the cytotoxic fluoronucleotides (FNuct). From these data, four parameters were measured: tumour pH and energy status (NTP/Pi) before treatment, total FU retention, and FU anabolism to FNuct (expressed as micromoles per gram per 2.5 h). In addition, tumour response was determined at 7 days post-treatment by measurement of the percentage of change in tumour weight and was classified according to standard oncological criteria as follows: progressive (P) for a > or =25% increase, remissive (R) for a > or =50% decrease or stable (S) for values lying between these two. RESULTS: Analysis of variance (ANOVA) for statistical assessment revealed that groups P, S and R were not distinguishable using the MRS parameters; although when S and R were combined as one group of non-progressive disease (NPD; n = 24), both the NTP/Pi ratio and the total FNuct formed were significantly greater (P = 0.04) than those observed in the P group (n = 5). Considering all 29 tumours, linear regression showed that there were positive significant correlations between the NTP/Pi ratio and (a) the percentage of response (P = 0.04), (b) the pre-treatment pH (P = 0.002) and (c) FU retention (P = 0.02), but not FNuct formation (P = 0.66). Unlike results reported in the clinic, the percentage of response and FU retention were neither significantly correlated (P = 0.22) nor associated when groups P and NPD were compared (P = 0.27, Fischer's exact test). FNuct, however, was significantly associated with response, as was the NTP/Pi ratio (P < or = 0.02). Combination of FNuct with the NTP/Pi ratio increased the significance of the association with response (P = 0.003, Fischer's exact test). CONCLUSIONS: Our results indicate that in this particular model the pre-treatment tumour NTP/Pi ratio was the best predictor of response to a bolus injection of FU, rather than FNuct formation or FU retention. An elevated NTP/Pi ratio could reflect a well-vascularised tumour with an improved capacity for energy-dependent FU uptake and metabolism to FNuct, suggesting that further investigation of this parameter could be an important line of research, which may aid the identification of tumours likely to be sensitive to FU chemotherapy in the clinic.

Analysis of Variance↗
PubMed2006

Clinical investigation: Utility of left ventricular end diastolic diameter in the prediction of susceptibility to ventricular tachyarrhythmias.

BACKGROUND: Prior studies have shown the utility of using both QRS duration and QT dispersion (QTd) as predictors of risk for ventricular tachyarrhythmias (VA). Lengthening of the QRS duration represents dyssynchrony of regional myocardial wall contraction, and increased QTd similarly represents variations in myocardial repolarization. We sought to examine the left ventricular end diastolic diameter (LVEDD) as a predictor of VA susceptibility. METHODS: Eighty-eight patients referred for electrophysiologic (EP) studies were evaluated. EP testing was performed using a standard protocol of up to three extrastimuli. QTd and QRS duration analyses were performed in a blinded manner. Values were defined as abnormal if QRS duration>120 ms, QTd>60 ms, and LVEDD>6 cm. RESULTS: Of 88 patients (65 males; 23 females; mean age 67+/-15 years), 33 were inducible by EP testing. Patients with either increased QRS duration or QTd are shown to be at greater risk for VA inducibility. LVEDD is a strong predictor of inducibility for VA (p<0.02 between inducible and non-inducible patients). LVEDD in combination with QRS duration and QTd, further strengthens predictability for VA (p<0.03 for QRS duration and p<0.02 for QTd) with a trend towards inducibility as each value increases. Combination of the three parameters of QRS duration, QTd, and LVEDD was 91% sensitive for the identification of those patients inducible for VA. CONCLUSION: The LVEDD is an echocardiographic value that strongly predicts VA inducibility, and when combined with QRS duration and QTd, identifies patients at higher risk for these tachyarrhythmias.

Aged↗
PubMed2006

Genetic data on 10 autosomal STR loci in the Bangladeshi population.

Allele frequencies of 10 autosomal short tandem repeat (STR) loci, D3S1358, vWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01 and FGA were determined in 211 unrelated Bangladeshi individual using AmpFLSTR SGM Plus PCR Amplification Kit. Statistical parameters of forensic importance, the power of discrimination (PD), observed and expected heterozygosity values (H), polymorphism information content (PIC), probability of match (PM), power of exclusion (PE) and typical paternity index (TPI) were calculated for the loci. These parameters indicated the usefulness of the loci in paternity testing and personal identification in the Bangladeshi population.

Bangladesh↗
PubMed2005

The relative contributions of different skin layers to the mechanical behavior of human skin in vivo using suction experiments.

Although the mechanical behavior of the top layer of the skin, the epidermis, is an important consideration in several clinical and cosmetic applications, there are few reported studies on this layer. The in vivo mechanical behavior of the upper skin layer (here defined as epidermis and papillar dermis) was characterized using a combined experimental and modeling approach. The work was based on the hypothesis that experiments with different length scales represent the mechanical behavior of different skin layers. Suction measurements with aperture diameters of 1, 2 and 6 mm were combined with ultrasound and optical coherence tomography to study the deformation of the skin layers. The experiments were simulated for small displacements with a two-layered finite element model representing the upper layer and the reticular dermis. An identification method compared the experimental and numerical results to identify the material parameters of the model. For one subject the whole parameter estimation procedure was completed, leading to a stiffness of C(10,ul) = 0.11 kPa for the top-layer and C(10,rd) = 0.16 MPa for the reticular dermis. This unexpected, extreme stiffness ratio of the material parameters let to convergence problems of the finite element software for most of the individuals.

Adult↗
PubMed2001

Quantitative structure-pharmacokinetic relationship modelling.

This article presents the current methods in quantitative structure-pharmacokinetic relationship (QSPkR) modelling along with examples using chemicals of toxicological significance. The common method involves: (i) collecting pharmacokinetic data or determining pharmacokinetic parameters (e.g. elimination half-life, volume of distribution) by fitting to experimental data; and (ii) associating them with the structural features of chemicals using a Free-Wilson model. Such QSPkRs have been developed for a few series of chemicals but their usefulness is limited to the exposure scenario and conditions under which the experimental data were originally collected. The alternative approach involves the development of quantitative structure-property relationship (QSPR) models for parameters, blood:air partition coefficient, tissue:blood partition coefficient, maximal velocity for metabolism and Michaelis affinity constant, of physiologically-based pharmacokinetic (PBPK) models which are useful for conducting species, route, dose and scenario extrapolations of the tissue dose of chemicals. Mechanistic QSPRs are available for predicting tissue:blood and blood:air partition coefficients from molecular structure information of chemicals, whereas such approaches are not currently available for hepatic metabolism parameters. However, at the present time, the pharmacokinetics of inhaled volatile organic chemicals can be simulated adequately by considering the physiological limits of the hepatic extraction ratio (0-1) and molecular structure-based estimates of partition coefficients in the PBPK model. This current state-of-the-art of structure-based modelling of pharmacokinetics will advance with the development of QSPRs for other chemical-specific parameters of PBPK models. Integrated QSPR-PBPK modelling should facilitate the identification of chemicals of a family that possess desired properties of bioaccumulation and blood concentration profile in both test animals and humans.

Animals↗
PubMed2006

Multiplex analysis of inflammatory signaling pathways using a high-content imaging system.

This chapter describes a robust high-content cellular screening assay to simultaneously analyze the spatiotemporal activation of three different kinase-associated signaling pathways involving NF-kappaB, JNK, and p38, all of which are closely implicated in proliferative and proinflammatory responses. Signal transduction is dependent on the translocation of NF-kappaB p65 and phosphorylated c-Jun and p38 from the cytosol to the nucleus, and fluorescent immunolabeling was used to monitor changes in their cellular distribution. Cellular screening, data acquisition, and data interpretation were conducted on the ArrayScan HCS Reader (Cellomics Inc., Pittsburgh, PA). Assay adaptation to various cellular systems is feasible when sufficient separation of the nuclear and cytosolic compartment can be achieved and if cell adhesion properties permit proper attachment to the culture plates. Substitution of NF-kappaB p65 and phosphorylated forms of c-Jun and p38 as targets to analyze other translocating components is possible and is limited primarily by antibody specificity and the risk of fluorescent bleed-through between emission channels. Because assay validity is particularly confounded by inadequate spectral separation of the detection dyes in multicolor labeling assays, means of eliminating or counterbalancing staining artifacts are illustrated. Also, protocol parameter settings important for imaging and image processing are described, including object identification, image exposure settings, separation of cytosolic and nuclear regions, number of cells sufficient for analysis, and the use of gating thresholds critical for cell sorting and subpopulation analysis. This assay is a useful tool to investigate the interplay between signaling pathways and the mode of action, potency, and selectivity of compound inhibition of specific target molecules in a cellular context.

Cell Nucleus↗