The resazurin assay, also known as the Alamar Blue assay, stands as a cornerstone technique in cell biology, microbiology, and drug development. It assesses the viability of cells through the ...conversion of resazurin into highly fluorescent resorufin. The resulting fluorescence intensity provides a reliable estimate of viable cell numbers. Cytotoxicity assays, such as the resazurin-based method, play a crucial role in the screening of potential drug candidates and in the assessment of pharmaceutical and chemical toxicity. In recent years, inconsistencies have arisen in pharmacogenomic studies, often due to poorly optimized laboratory protocols. These inconsistencies hinder progress in understanding how substances affect cell health, leading to unreliable findings. Thus, the need for standardized and rigorously optimized protocols is evident to ensure consistent and accurate results in cytotoxicity studies. This manuscript describes a standardized procedure for optimizing resazurin-based viability assays to improve the reliability of cytotoxicity data. This optimization approach focuses on critical experimental parameters and data quality, aiming to achieve a level of measurement imprecision of less than 20%. In conclusion, to address the critical issues of reproducibility and reliability, protocol standardization, such as the one described in this manuscript, can greatly enhance the credibility of cytotoxicity studies, ultimately advancing drug safety assessments.
Aim
To evaluate and quantify the intervention fidelity of a symptom management protocol through implementation of a scorecard, using an exemplar study of caregiver‐delivered reflexology for people ...with breast cancer.
Background
Studies on caregiver‐delivered symptom management interventions seldom include adequate information on protocol fidelity, contributing to potentially suboptimal provision of the therapeutic intervention, hindering reproducibility and generalizability of the results.
Design
Fidelity assessment of a 4‐week intervention protocol in a randomized controlled trial (RCT) with data collection between 2012 ‐ 2016.
Methods
The National Institutes of Health Behaviour Change Consortium (NIH‐BCC) conceptual model for intervention fidelity guided the study. The five NIH‐BCC fidelity elements are: (1) dose; (2) provider training; (3) intervention delivery; (4) intervention receipt; and (5) enactment. To illustrate the elements, an intervention protocol was deconstructed and each element quantified using a newly developed fidelity scorecard.
Results
Mean scores and frequency distributions were derived for the scorecard elements. For dose, the mean number of sessions was 4·4, 96% used the correct intervention duration and 29% had 4 weeks with at least one session. Provider training was achieved at 80% of the maximum score, intervention delivery was 96%, intervention receipt was 99% and enactment indicated moderate adoption at 3·8 sessions per patient. The sample mean score was 15·4 out of 16, indicating the high overall fidelity.
Conclusion
Research findings that include description of how fidelity is both addressed and evaluated are necessary for clinical translation. Clinicians can confidently recommend symptom management strategies to patients and caregivers when fidelity standards are explicitly reported and measured.
This study aimed to investigate the influence of immersion duration and the type of immersion solution on the outcome of push-out bond strength (POBS) tests. Root canals of 120 straight single-rooted ...teeth were instrumented to a diameter of 1.5 mm and irrigated with 5 mL of 3% NaOCl. Four horizontal slices with a thickness of 1 mm were cut, representing the mid-portion of the root. The specimens (n = 480) were irrigated with 17% ethylenediaminetetraacetic acid(EDTA) for 60 seconds, then twice with distilled water (DW) for 30 s each. The canals were filled with either AH Plus (Dentsply Sirona, Konstanz, Germany) or BioRoot RCS (Septodont, St. Maur-des-Fossés, France) (n = 240). Separated into four groups per type of sealer (n = 60), the specimens were incubated at 37 °C covered with gauze moistened in DW or phosphate-buffered saline (PBS) for either one or eight weeks. Dislodgement resistance was measured and POBS was calculated. Statistical analysis was performed using the analysis of variance (ANOVA) test and the Student-Newman-Keuls test (
= 0.05). AH Plus showed higher POBS when stored in PBS compared to DW, irrespective of the incubation period (
< 0.05). BioRoot RCS displayed higher POBS when stored in DW compared to PBS after eight weeks of incubation (
< 0.05). No difference was found after one week of incubation (
> 0.05). Irrespective of the sealer or the immersion solution, POBS decreased from one week to eight weeks (
< 0.05). Mixed failure modes were found in all groups irrespective of sealer, immersion medium, or immersion period. POBS decreased after a longer incubation time in both immersion solutions. Duration of immersion and the type of immersion solution had a significant impact on the outcome of the POBS testing protocol.
Standardization is crucial when culturing cells including human embryonic stem cells (hESCs) which are valuable for therapy development and disease modeling. Inherent issues regarding reproducibility ...of protocols are problematic as they hinder translation to good manufacturing practice (GMP), thus reducing clinical efficacy and uptake. Pluripotent cultures require standardization to ensure that input material is consistent prior to differentiation, as inconsistency of input cells creates end-product variation. To improve protocols, developers first must understand the cells they are working with and their related culture dynamics. This innovative work highlights key conditions required for optimized and cost-effective bioprocesses compared to generic protocols typically implemented. This entailed investigating conditions affecting growth, metabolism, and phenotype dynamics to ensure cell quality is appropriate for use. Results revealed critical process parameters (CPPs) including feeding regime and seeding density impact critical quality attributes (CQAs) including specific metabolic rate (SMR) and specific growth rate (SGR). This implied that process understanding, and control is essential to maintain key cell characteristics, reduce process variation and retain CQAs. Examination of cell dynamics and CPPs permitted the formation of a defined protocol for culturing H9 hESCs. The authors recommend that H9 seeding densities of 20,000 cells/cm
2
, four-day cultures or three-day cultures following a recovery passage from cryopreservation and 100% medium exchange after 48 hours are optimal. These parameters gave ~SGR of 0.018 hour
−1
± 1.5x10
−3
over three days and cell viabilities ≥95%±0.4, while producing cells which highly expressed pluripotent and proliferation markers, Oct3/4 (>99% positive) and Ki-67 (>99% positive).
With the advent of novel strategies to induce tolerance in autoimmune and autoimmune-like conditions, clinical trials of antigen-specific tolerizing immunotherapy have become a reality. Besides ...safety, it will be essential to gather mechanistic data on responding CD4
T cells to assess the effects of various immunomodulatory approaches in early-phase trials. Peptide-MHC class II (pMHCII) multimers are an ideal tool for monitoring antigen-specific CD4
T cell responses in unmanipulated cells directly
. Various protocols have been published but there are reagent and assay limitations across laboratories that could hinder their global application to immune monitoring. In this methodological analysis, we compare protocols and test available reagents to identify sources of variability and to determine the limitations of the tetramer binding assay. We describe a robust pMHCII flow cytometry-based assay to quantify and phenotype antigen-specific CD4
T cells directly
from frozen peripheral blood mononuclear cell samples, which we suggest should be tested across various laboratories to standardize immune-monitoring results.
Through this investigation we developed a methodology to evaluate and standardize CT image quality from routine abdomen protocols across different manufacturers and models. The influence of ...manufacturer-specific automated exposure control systems on image quality was directly assessed to standardize performance across a range of patient sizes. We evaluated 16 CT scanners across our health system, including Siemens, GE, and Toshiba models. Using each practice's routine abdomen protocol, we measured spatial resolution, image noise, and scanner radiation output (CTDIvol). Axial and in-plane spatial resolutions were assessed through slice sensitivity profile (SSP) and modulation transfer function (MTF) measurements, respectively. Image noise and CTDIvol values were obtained for three different phantom sizes. SSP measurements demonstrated a bimodal distribution in slice widths: an average of 6.2 ± 0.2 mm using GE's 'Plus' mode reconstruction setting and 5.0 ± 0.1 mm for all other scanners. MTF curves were similar for all scanners. Average spatial frequencies at 50%, 10%, and 2% MTF values were 3.24 ± 0.37, 6.20 ± 0.34, and 7.84 ± 0.70 lp cm(-1), respectively. For all phantom sizes, image noise and CTDIvol varied considerably: 6.5-13.3 HU (noise) and 4.8-13.3 mGy (CTDIvol) for the smallest phantom; 9.1-18.4 HU and 9.3-28.8 mGy for the medium phantom; and 7.8-23.4 HU and 16.0-48.1 mGy for the largest phantom. Using these measurements and benchmark SSP, MTF, and image noise targets, CT image quality can be standardized across a range of patient sizes.
Down syndrome (DS), caused by trisomy of chromosome 21 (Hsa21), results in a spectrum of phenotypes including learning and memory deficits, motor dysfunction and social constrains. The regions on ...Hsa21 are conserved with their synteny on mouse chromosome 10, 16 and 17. To date, a wide range of mouse models has been developed to determine genotype-phenotype relationships and identity of the causative dosage-sensitive genes. However, the comparison of behavioral results is not obvious due to the lack of consistency in the genetics background, housing conditions and behavioral protocols used. There is a growing need to standardize some of the classical behavioral test, include automated behavioral phenotyping and sophisticated analysis techniques and move through ethologically inspired tests. Here we present an overview of the status of behavioral phenotyping of DS murine models and the limitations and possibilities to improve their characterization to address genotype-phenotype relationships for understanding the pathophysiology of DS.
During the last years, the research on extracellular vesicles (EVs) has raised giving new insights into pathophysiology of several diseases. EVs are membrane-bound particles secreted by almost all ...cell types. Depending on their biogenesis and size they include exosomes, microparticles / microvesicles and apoptotic bodies. Characteristically, EVs carry markers from the source cell membrane and contain genetic material, lipids and proteins inside. They are known to play a role in cell-to-cell communication and to produce genotypic and phenotypic modifications in the target cell including: antigen presentation, apoptosis induction, cellular activation, inhibition or differentiation. In particular, increasing concentrations of EVs have been found in many diseases such as cancer, autoimmune and cardiovascular diseases, among others. Most of the studies in EVs are focused on the characterization of EVs compounds, identifying mechanism of action, their potential use as biomarkers, and few of them investigate a therapeutic usage. However, there are some issues to be achieved on the path to their clinical application. This research topic offers a common place to discuss current and novel clinical applications of EVs pointing on future directions. We encouraged the submission of original articles, reviews, hypothesis, controversies, future perspectives and personal viewpoints on the following topics of interest, but not limited to: • Contribution of EVs to better understand the pathology of immunological diseases. • Standardization of isolation and quantification protocols in the daily clinical practice. • Possible applications of EVs as clinical biomarkers (diagnostic, prognostic and evolution marker). • Therapeutic role of EVs being vehicles of specific cargo: current clinical trials? • Novel immunological functions of EVs.
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