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Immune responses of intact and embryonically enucleated frogs to self-lens antigens.

Embryonically enucleated frogs (Xenopus) will tolerate an isogeneic eye implanted during adult life although their immune system has never been previously exposed to eye-specific Ag. We hypothesized that such self-implants survive because they induce tolerance to the eye-specific Ag. We have developed evidence to support this hypothesis by studying the responses of embryonically enucleated or intact frogs, with or without eye implants, to self-lens proteins. After immunization with a low concentration of bovine lens proteins, spleen cells from all intact and all embryonically enucleated frogs displayed significant in vitro proliferation against the Ag. All animals in both groups of frogs also produced high titered xenoantibodies. After immunization with a comparable preparation and concentration of self-lens Ag, splenocytes from only some of the embryonically enucleated and intact frogs showed significant proliferation, and fewer of these frogs produced antibody. When the immunization protocol and Ag concentration were modified to evoke consistent anti-self-lens proliferative and antibody responses from enucleated frogs, intact frogs responded equally well. Because there were no significant differences in the magnitude of the responses, and the percent of enucleated and intact frogs responding did not differ, we conclude that there is little or no immunologic tolerance to self-lens Ag in intact frogs. When either embryonically enucleated or intact frogs were heterotopically implanted with isogeneic eyes and then immunized with self-lens Ag or foreign lens Ag using the optimal immunization protocol, the Ag-specific proliferative responses of their lymphocytes were significantly reduced after immunization with self-, but not foreign Ag. This argues that embryonically enucleated frogs can become tolerant of the organ-specific Ag of the lens, even in adult life.

Animals↗

A practical approach to the first iteration in the optimization of radiation dose and image quality in CT: estimates of the collective dose savings achieved.

CT is a diagnostic imaging modality giving higher patient dose in comparison with other radiological procedures. It contributed an estimated 20% to the collective effective dose to the UK population from medical X-rays in 1990, rising to an estimated 40% in 1999. Tremendous national effort has been expended in reducing patient doses from other radiological procedures with considerable success, but much of the collective dose reduction achieved has been offset by a corresponding increase in the collective dose from CT. Over a period of about 2 years, following the installation of a helical CT scanner, CT scan parameters in this hospital have been adjusted with the aim of working towards optimization of image quality and patient dose. A 33% reduction in annual collective effective dose has been achieved, from about 16.5 manSv to 11 manSv. However, despite this dose reduction, the annual collective effective dose to our sub-population is 2.8 times the value 9 years ago. The increase is almost entirely the result of an increased application of CT; 6149 examinations per annum in 1999 compared with 2210 in 1991. The crucial importance of reducing patient doses from this modality is discussed. Indicative effective doses and image noise values are presented for examination protocols approaching optimization.

Humans↗

Comparative study of cell culture and purification methods to obtain highly enriched cultures of proliferating adult rat Schwann cells.

We present here a fast protocol that could be used to obtain highly purified cultures of maximal proliferating adult rat Schwann cells. These adult rat Schwann cells can be transfected in a nonbiological way using the physical transfection method of electroporation. Schwann cells are decisive in recovery of peripheral nerves after injury. In a clinical context, the use of enriched adult Schwann cells is necessary for autologous cell transplantation within nerve transplants for peripheral nerve repair. Different parameters such as tissue preparation, culture conditions, and protocols for enrichment, elevation of proliferation rates, and transfection were evaluated in cell cultures harvested from adult rat peripheral nerves. Cell preparation from in vivo predegenerated adult rat sciatic nerves combined with the use of melanocyte growth medium supplemented with forskolin, fibroblast growth factor (FGF)-2, and pituitary extract as a selective, serum-free culture medium, with a secondary cell-enrichment step using specific detachment, resulted in highly enriched cultures of adult rat Schwann cells (>90%) with enhanced proliferation rates (>or=40%). About 20% of these adult Schwann cells could be modified genetically using an optimized electroporation protocol.

Animals↗

Human amnion mesenchyme cells express phenotypes of neuroglial progenitor cells.

Previous studies from our laboratory showed that human amnion epithelial cells (AECs) have multiple functions, such as synthesis and release of catecholamines, acetylcholine, neurotrophic factors, activin, and noggin. In this study, we investigated the identity of neural progenitor cells in human amnion mesenchyme cells (AMCs), which lie immediately adjacent to the AECs. Cryostat sections revealed that vimentin expression was detected in the AMCs and CK19 in AECs. Vimentin-positive cells made up 97.5% of total cells tested in cultured AMCs. Interestingly, 3.6% of total AMCs expressed the phenotype CK19+/vimentin+, indicating coexpression of epithelial and mesenchyme cell markers. In culturing with bromodeoxyuridine (BrdU) for 24 hr, 66-82% of cells were found to be BrdU positive, suggesting that they have proliferating potency. By using RT-PCR, AMCs express mRNA of nestin and Musashi1. With a neural cell differentiating protocol, cell bodies extended long bipolar or complex multipolar processes. Nestin (87.7% of total cells tested) and Musashi1 (93.1%) were expressed in undifferentiated cells, and their positively stained cells increased in number slightly after induction. Undifferentiated cells were stained by anti-Tuj1 and NF-M, and their positively stained cells increased significantly in number after induction, to 72.8% and 46.0%, respectively. Meanwhile, glial fibrillary acidic protein-positive cells increased from 25.4% to 43.2% after induction. These studies demonstrate that AMCs have phenotypes of neuroglial progenitor cells and can be differentiated into neuroglial phenotypes by optimal differentiation protocol. Eventually, AMC-derived stem cells may be a favorable cell vehicle in regenerative medicine.

Amnion↗

Quantification of signal changes in gradient recalled echo FMRI.

Understanding and quantifying the various contributions to functional magnetic resonance imaging (FMRI) signal changes in activated cortical areas is paramount for a clinical application of brain mapping by FMRI. Therefore, all significant contributions to FMRI signal changes, both extra- and intravascular, from macrovessels down to the capillary network, should be taken into account. We present a gradient-recalled-echo FMRI model based on in-flow effects described by the Bloch equations, adding susceptibility effects empirically via T2* differences measured in vitro in human blood samples. Results of these calculations (by systematically varying alpha, echo time (TE), repetition time (TR), as well as blood velocity and T2* upon stimulation) may be used to (a) simulate functional MRI experiments with different measurement protocols and (b) estimate realistic values for important anatomical and physiological details that influence local signal changes in FMRI (i.e., size and distribution of vessels, effective relaxation times of blood, etc.). The excellent agreement between our model calculations and experimental results from conventional gradient recalled echo fMRI in vivo suggests a significant contribution from very slow flow and oxygenation changes, predominantly in small vessels (vblood = 1-4 mm/s). The actual contribution of T1- and T2-related effects is strongly dependent on sequence design and actual sequence parameters used. Thus, the model simulations presented may also be used to optimize measurement protocols for investigating various neurophysiological phenomena.

Adolescent↗

Transgenic tumor models for carcinogen identification: the heterozygous Trp53-deficient and RasH2 mouse lines.

Genetically altered mouse models (GAMM) for human cancers have been critical to the investigation and characterization of oncogene and tumor suppressor gene expression and function and the associated cancer phenotype. Similarly, several of the mouse models with defined genetic alterations have shown promise for identification of potential human carcinogens and investigation of mechanisms of carcinogen-gene interactions and tumorigenesis. In particular, both the B6.129N5-Trp53 mouse, heterozygous for a p53 null allele, and the CB6F1-RasH2 mouse, hemizygous for the human H-ras transgene, have been extensively investigated. Using 26-week exposure protocols at or approaching the maximum tolerated dose, the summary results to date indicate the potential for GAMM to identify and, possibly, classify chemicals of potential risk to humans using short-term carcinogenicity experiments. This IWGT session focused on: (1) the development of recommendations for genetic/molecular characterization required in animals, tissues, and tumors before and after treatment for identification of presumptive human carcinogens based on the current state of knowledge, (2) identification of data gaps in our current state of knowledge, and (3) development of recommendations for research strategies for further development of our knowledge base of these particular models. By optimization of protocols and identification of significant outcomes and responses to chemical exposure in appropriate short-term mechanism-based genetically altered rodent models, strategies for prevention and intervention may be developed and employed to the benefit of public health.

Animals↗

Screening for fever by remote-sensing infrared thermographic camera.

BACKGROUND: Following the severe acute respiratory syndrome (SARS) outbreak, remote-sensing infrared thermography (IRT) has been advocated as a possible means of screening for fever in travelers at airports and border crossings, but its applicability has not been established. We therefore set out to evaluate (1) the feasibility of IRT imaging to identify subjects with fever, and (2) the optimal instrumental configuration and validity for such testing. METHODS: Over a 20-day inclusive period, 176 subjects (49 hospital inpatients without SARS or suspected SARS, 99 health clinic attendees and 28 healthy volunteers) were recruited. Remotely sensed IRT readings were obtained from various parts of the front and side of the face (at distances of 1.5 and 0.5 m), and compared to concurrently determined body temperature measurements using conventional means (aural tympanic IRT and oral mercury thermometry). The resulting data were submitted to linear regression/correlation and sensitivity analyses. All recruits gave prior informed consent and our Faculty Institutional Review Board approved the protocol. RESULTS: Optimal correlations were found between conventionally measured body temperatures and IRT readings from (1) the front of the face at 1.5m with the mouth open (r=0.80), (2) the ear at 0.5 m (r=0.79), and (3) the side of the face at 1.5m (r=0.76). Average IRT readings from the forehead and elsewhere were 1 degrees C to 2 degrees C lower and correlated less well. Ear IRT readings at 0.5 m yielded the narrowest confidence intervals and could be used to predict conventional body temperature readings of < or = 38 degrees C with a sensitivity and specificity of 83% and 88% respectively. CONCLUSIONS: IRT readings from the side of the face, especially from the ear at 0.5 m, yielded the most reliable, precise and consistent estimates of conventionally determined body temperatures. Our results have important implications for walk-through IRT scanning/screening systems at airports and border crossings, particularly as the point prevalence of fever in such subjects would be very low.

Adolescent↗

Distribution of renal lesions in idiopathic systemic vasculitis: A three-dimensional analysis of 87 glomeruli.

Extracapillary proliferation and fibrinoid necrosis are the main diagnostic glomerular lesions in renal biopsy specimens of patients with idiopathic systemic vasculitis. Neither the incidence nor the correlation between extracapillary proliferation and fibrinoid necrosis in renal biopsy specimens from patients with systemic vasculitis has been systematically evaluated. By means of a three-dimensional analysis, we made a topographic reconstruction of the distribution of extracapillary proliferation and fibrinoid necrosis in affected glomeruli and tested different biopsy-processing protocols to optimize histopathologic analysis in clinical practice. Paraffin blocks of renal biopsy specimens from six patients diagnosed with systemic vasculitis were completely and serially sectioned in 2-microm thick sections and stained with the Gomori trichrome method. Glomeruli were scored per section for the presence of fibrinoid necrosis and extracapillary proliferation. Subsequently, a three-dimensional reconstruction was obtained for 87 glomeruli. In only one glomerulus did fibrinoid necrosis occur without extracapillary proliferation; in 51%, a combination of the two lesions was found; in 22%, extracapillary proliferation occurred in the absence of fibrinoid necrosis; and 26% did not show either lesion. Using the standard protocol from our department (ie, evaluation of 20 consecutive sections in various stainings), the chance of finding extracapillary proliferation was 100% and that of finding fibrinoid necrosis was 73%. If 5 sections stained with the Gomori trichrome were added, the latter percentage increased to 86%. Using skip-serial sections, even better results (87% to 92%) were obtained, with four skips as the best option (92%). In conclusion, our finding that fibrinoid necrosis rarely occurs in the absence of extracapillary proliferation may imply that both lesions are etiologically related. In addition, our observations indicate that the incidence of fibrinoid necrosis may be underestimated in clinical practice, depending on the number of sections evaluated.

Biopsy↗

A strategy for optimizing quality and quantity of DNA extracted from soil.

The efficiency of a bead beating method was studied in detail with regard to a variety of factors including beating time and speed, volume and temperature of the buffer, as well as amount and type of beads employed. The results presented here reveal that all of these parameters have a significant effect on yield and quality of DNA extracted from soils. Precise adjustment of extraction conditions allows for significantly higher yields of high quality DNA from soils than previously reported. We further evaluated the effect of the extraction conditions on the apparent soil microbial community structures, as observed by polymerase chain reaction (PCR) and RFLP. Differences in the fingerprints of DNA extracted under different conditions suggest that results could be biased when using gentle extraction procedures. Based on multiple subsequent extractions using very harsh extraction conditions, we propose a protocol for the quantification of the total DNA content in soils. Extractions from six soils of different texture and chemical characteristics with selected bead beating protocols revealed that the quality (fragment size and purity) of the extracted DNA was generally very good, but also depended on the soil characteristics. While a single, general protocol for optimal DNA recovery from all soils cannot be given, this study provides detailed guidelines on how to optimize the general method to obtain optimal DNA from individual soils.

Buffers↗

The short-term storage and cryopreservation of spermatozoa from hylid and myobatrachid frogs.

The short-term storage (at 0 degrees C) and cryopreservation of spermatozoa may be useful for providing gametes for fertilisations performed in programmes for the conservation and management of endangered amphibians. The current study was undertaken to examine the applicability of amphibian spermatozoa storage protocols developed with the cane toad (Bufo marinus) to a wider range of amphibian species, with a view to ultimately using these protocols for endangered species. In Australia, at least 29 species of recently extinct or endangered frogs are from the families the Myobatrachidae and the Hylidae. This study investigated the applicability of short-term storage and cryopreservation protocols developed for cane toad (Bufo marinus) spermatozoa to those of hylid and myobatrachid species. Storage of spermatozoa in intact testes or in suspensions for six days at 0 degrees C showed spermatozoa maintained higher motility in suspensions than those in testes, and hylid spermatozoa maintained greater motility than myobatrachid spermatozoa. However, the protocols for optimal storage at 0 degrees C varied with testis size when spermatozoa were stored in whole testes. Spermatozoa from 13 frog species representing both families were cryopreserved using sucrose as diluent with Me(2)SO or glycerol as cryoprotectants. After cryopreservation hylid spermatozoa showed a greater recovery than myobatrachid spermatozoa and Me(2)SO provided higher recovery than glycerol. The freeze-thaw recovery of spermatozoa was independent of testes weight of the species studied. These results show spermatozoa from the Hylidae and Myobatrachidae may be stored both in the short-term (at 0 degrees C) and long-term by cryopreservation using protocols established for B. marinus.

Animals↗

[Problems in inducing pulmonary maturation in relation to premature delivery].

At presents efforts are made to find valid early markers of premature delivery and if it is inevitable to use an optimal therapeutic protocol for induction of pulmonary maturity which may lead to reduction of early neonatal mortality and morbidity (intraventricular-periventricular haemorrhage, necrotizing enterocolitis, bronchopulmonary dysplasia, air-leak syndrome, respiratory distress syndrome--RDS). Hitherto used corticoid monotherapy reduced the incidence of RDS in neonates with a birth weight lower than 1500 g but the incidence is still relatively high and RDS is still one of the main causes of death of immature neonates. Even the use of thyrotropic hormones did not reduce its incidence. Another possible therapeutic approach is ambroxol administration in combination with corticoids. In the presented review the authors discuss the problem of optimal induction of pulmonary maturity in relation to premature delivery.

Ambroxol↗

Experiences with gadodiamide, a non-ionic contrast agent, in MRI of brain metastases.

29 patients suffering from brain metastases were studied with a single (0.1 mmol/kg body weight) and triple dose (0.3 mmol/kg) of gadodiamide, a non-ionic MR contrast agent. The study was performed using an extended imaging protocol with application of T1-weighted images in three orientations. Number of definite and suspected metastases, lesion contrast, edema delineation, and flow artefacts were evaluated. Most of the definite metastases were found in triple dose images. However, the total lesion number could only be determined by looking on all sequences as a whole. Furthermore, the number of suspected metastases could be significantly reduced with triple dose images, but once more all sequences were needed to do so. Flow artefacts were significantly increased by triple dose, whereby lesion contrast and edema delineation was considerably improved. The conclusion is that the combination of high dose application of the contrast agent and an optimized imaging protocol facilitates sufficient imaging of brain metastases. This is important for therapeutical considerations, because patients with more than 1-2 metastases will be treated primarily with radiation therapy instead of neurosurgery.

Brain Neoplasms↗

Practical strategies for performance optimization of the enhanced gen-probe amplified mycobacterium tuberculosis direct test.

The enhanced Gen-Probe Amplified Mycobacterium Tuberculosis Direct (MTD) test was evaluated using a combined set of 338 acid-fast smear-positive and smear-negative, respiratory and nonrespiratory clinical specimens received by the Massachusetts State Tuberculosis Laboratory from September 1999 through March 2002. Microbiological culture was used as the reference method; therefore, the sensitivity and specificity of the MTD test were calculated for culture-positive specimens only. The initial assessment indicated that the overall sensitivity, specificity, and positive and negative predictive values of the MTD test for all specimens grouped together were 62, 98, 99, and 68%, respectively. A detailed discrepancy analysis revealed that two major factors causing negative MTD results in specimens that were culture positive for M. tuberculosis complex were patient treatment with antituberculosis drugs prior to testing and the presence of inhibitory substances in the specimen. Based on these findings, a protocol for optimizing MTD test performance in this setting is proposed in which (i) specimens from patients taking antituberculosis medications are excluded from testing and (ii) all initially MTD-negative or MTD-equivocal specimens are subjected to testing for inhibitors. If this strategy was followed, the MTD test sensitivity would be at least 91%, a significant improvement over the initial sensitivity of 62%. Accordingly, the negative predictive value would increase from 68 to 91%.

Culture Media↗

Light and electron microscopic in situ hybridization: non-radioactive labeling and detection, double hybridization, and combined hybridization-immunocytochemistry.

We performed light and electron microscopic in situ hybridization, according to the same protocol and without pretreatment of sections, on Lowicryl- and LR Gold-embedded cells. Digoxigenin (DIG)- or biotin-labeled riboprobes were visualized by direct or indirect immunodetection using commercially available gold-antibody conjugates with 0.8-10-nm gold grains. At the ultrastructural level, the main findings were that DIG-labeled probes gave a slightly higher labeling intensity (grains per signal) than biotin. The direct detection method produced a more compact signal, which led to better resolution at medium and high magnifications. Labeling intensities of all gold grain sizes were essentially equal. Grain sizes of 5 nm and larger were highly preferable because available enhancement methods are unsatisfactory for ultrasmall grains. The optimized immunodetection protocols are suitable for double hybridization with two different probes and for combined hybridization and immunocytochemistry.

Antibodies↗

A versatile strategy for preimplantation genetic diagnosis of haemophilia A based on F8-gene sequencing.

Preimplantation genetic diagnosis (PGD) of hemophilia A (HA) and other X-linked diseases through sex selection implies that male embryos will be systematically discarded, even though 50% are unaffected. The objective of the present work was to develop a PGD protocol for direct mutation identification that could be applied to first polar bodies (1PBs) in several HA clinical cases. Single buccal cells from controls and patients, and 1PBs were subjected to primer extension preamplification (PEP) PCR followed by amplification of F8 gene coding and intronic flanking regions, and direct sequencing. Moreover, multiplex fluorescent amplification of four short tandem repeats was adapted to a single cell preamplification in order to rule out contamination and allele drop-out, and for confirmatory indirect diagnosis. A couple at risk of HA transmission, with a familial mutation characterized as a 41-bp duplication in exon 14 of the F8 gene, was selected for the first clinical study. After optimizing the protocol, the complete F8 gene coding sequence was obtained from single cells to demonstrate the sensitivity of our methodology although in any clinical case only the relevant region, not the whole gene, must be amplified. The woman enrolled in the first clinical case has completed the first in-vitro fertilization cycle, and seven oocytes were analyzed with concordant results by both linkage analysis and direct sequencing method. Only one oocyte, among those diagnosed as mutation free, developed to embryo at day 3. It was transferred but pregnancy was not achieved. This PGD procedure enables non-affected and noncarrier embryo selection in families with any point or small-range mutation in the F8 gene, without the need for further custom-made modifications.

DNA Mutational Analysis↗

Standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval technique: from experiments to hypothesis.

From a practical point of view, one of the most difficult issues in the standardization of IHC for FFPE tissue is the adverse influence of formalin upon antigenicity, as well as the great variation in fixation/processing procedures. Based on previous study, an additional study using four markers demonstrated the potential for obtaining equivalent IHC staining among FFPE tissue sections with periods of formalin fixation ranging from 6 hr to 30 days. On this basis, the following hypothesis is proposed. "The use of optimized AR protocols permits retrieval of specific proteins (antigens) from FFPE tissues to a defined and reproducible degree (expressed as R%), with reference to the amount of protein present in the original fresh/unfixed tissue". This hypothesis may also be presented mathematically: the protein amount in a fresh cell/tissue, expressed as Pf, produces an IHC signal in fresh tissue of integral(Pf). When the identical IHC staining plus AR treatment is applied to a FFPE tissue section, the IHC signal may be represented as integral (Pffpe). The degree of retrieval after AR (R%) is calculated as follows: R% = integral (Pffpe)/ integral (Pf) x 100%. The amount of protein in the FFPE tissue may then be derived as follows: Pffpe = Pf x R%. In a situation where optimized AR is 100% effective, the IHC signal would then be of equal strength in fresh tissue and FFPE tissue, and Pffpe= Pf. Further studies are designed to test the limitations of the proposed hypothesis.

Antigens↗

Radiation dose and image quality in diagnostic radiology. Optimization of the dose-image quality relationship with clinical experience from scoliosis radiography, coronary intervention and a flat-panel digital detector.

UNLABELLED: X-rays are known to cause malignancies, skin damage and other side effects and they are thus potentially dangerous. Therefore, it is essential and in fact mandatory to reduce the radiation dose in diagnostic radiology as far as possible. This is also known as the ALARA (as low as reasonably achievable) principle. However, the dose is linked to image quality and the image quality may not be lowered so far that it jeopardizes the diagnostic outcome of a radiographic procedure. The process of reaching this balance between dose and image quality is called optimization. The aim of this thesis was to propose and evaluate methods for optimizing the radiation dose-image quality relationship in diagnostic radiography with a focus on clinical usefulness. The work was performed in three main parts. OPTIMIZATION OF SCOLIOSIS RADIOGRAPHY: In the first part, two recently developed methods for digital scoliosis radiography (digital exposure and pulse fluoroscopy) were evaluated and compared to the standard screen-film method. Radiation dose was measured as kerma area-product (KAP), entrance surface dose (ESD) and effective dose; image quality was assessed with a contrast-detail phantom and through visual grading analysis. Accuracy in angle measurements was also evaluated. The radiation dose for digital exposure was nearly twice as high as the screen-film method at a comparable image quality while the dose for pulsed fluoroscopy was very low but with a considerably lower image quality. The variability in angle measurements was sufficiently low for all methods. Then, the digital exposure protocol was optimized to a considerably lower dose with a slightly lower image quality compared to the baseline. FLAT-PANEL DETECTOR: In the second part, an amorphous-silicon direct digital flat-panel detector was evaluated using a contrast-detail phantom, measuring dose as entrance dose. The flat-panel detector yielded a superior image quality at a lower dose than both storage phosphor plates and screen-film. Equivalent image quality compared to storage phosphor plates was reached at about one-third of the dose. OPTIMIZATION OF PERCUTANEOUS CORONARY INTERVENTION (PCI): In the third part, influence of various settings on radiation dose and image quality in coronary catheterisation and PCI was investigated. Based on these findings, the dose rate for fluoroscopy was reduced to one-third. The dose reduction was evaluated in a clinical series of 154 PCI procedures before and 138 after the optimization. Through this optimization, the total KAP was significantly reduced to two-thirds of the original value. IN SUMMARY: This thesis indicates the possibility of dose reduction in diagnostic radiology through optimization of the radiographic process.

Coronary Angiography↗

Miniaturization of a functional transcription assay in yeast (human progesterone receptor) in the 384- and 1536-well plate format.

Miniaturization of high throughput screening assays to high-density microplate formats (384 or 1536 wells) is currently the focus of research activity in modern drug discovery facilities. In this article, we describe the adaptation of a fluorescence-based functional transcription assay in yeast for assessing modulators of human progesterone receptor to the 384- and 1536-well microplate format, comparing the experimental results to those obtained in the well-established 96-well format. The experiences gained from the optimization of the liquid-handling procedures and the miniaturization of an enzyme assay (beta-galactosidase) were implemented. Thus optimized pipetting protocols were developed to perform a reporter gene assay in yeast in microplate formats of higher density. In the functional transcription assay in yeast, the reporter gene expression showed the expected dependence on the ligand's dose and affinity in principle in all three microplate formats. For the first time, this assay system has been established in the 1536-well microplate format using CyBi-Well 96/384/1536 as the liquid-handling unit. The comparison of the signal:background ratios showed a lower sensitivity of the assay in the microplate formats of higher density. This study is an example of a successful miniaturization of a yeast cell-based assay to high-density plate formats on the basis of a careful adaptation procedure and optimized liquid-handling conditions.

Cloning, Molecular↗