Methods and Protocols最新文献

Accurate quantification of immune cell subpopulations is essential for understanding immune responses in research and clinical settings. Flow cytometry (FC) is widely used for immune cell phenotyping, providing rapid and quantitative single-cell resolution. However, tissue-based pathological assessment offers additional spatial and morphological context that is often necessary for a comprehensive understanding of immune cell distribution. Traditionally, these methods are applied separately to different specimens, limiting direct comparative analysis. Here, we describe a simple combined approach to immune cell quantification that integrates both FC and pathology analysis within the same tissue specimen of colon biopsies. Tissue samples were divided into two portions: one processed into a single-cell suspension for FC-based characterization of CD45⁺, CD3⁺, CD4⁺, and CD8⁺ T cells and another formalin-fixed, paraffin-embedded (FFPE), and analyzed with hematoxylin and eosin (H&E) for eosinophils and immunohistochemistry (IHC) for CD4 and CD8. A pilot analysis of 10 samples shows high concordance of the results taken from the two methods, allowing for cross-validation between methodologies and improved diagnostic accuracy. This proof-of-principle study demonstrates the feasibility of an integrated workflow that combines FC and pathology for immune cell quantification, which provides assessment of immune cell populations from the limited material of intestinal biopsies with potential for improved diagnostic accuracy.

Background: Extensive skin injuries from severe burns or chronic non-healing ulcers overwhelm the body's natural repair mechanisms, while current therapeutic approaches relying on autologous skin grafting are limited by donor site availability. Three-dimensional epithelial spheroid cultures enhance stem cell regenerative potential, but standardized comparative methodologies are lacking. Methods: We established a comprehensive framework comparing scaffold-free and scaffold-based epithelial spheroid systems using HaCaT keratinocytes. High-throughput approaches utilized BioFloat and ELPLASIA 96-well platforms, while low-throughput 6-well ULA plates generated heterogeneous populations (holospheres, merospheres, paraspheres). Scaffold-based studies embedded spheroids in Matrigel to evaluate outgrowth capacity. ROCK1 inhibitor treatment was assessed for stemness enhancement. Results: High-throughput systems generated uniform spheroids with high reproducibility and consistent circularity. Low-throughput cultures produced heterogeneous populations with distinct size distributions (holospheres: 408.7 μm², merospheres: 99 μm², paraspheres: 14.1 μm²). In Matrigel scaffolds, merospheres and paraspheres migrated outward, forming epithelial sheets, while holospheres remained intact as BMI-1+ stem cell reservoirs. ROCK1 inhibition enhanced holosphere formation, preserved stemness markers, and reduced premature differentiation. Conclusions: This standardized toolbox demonstrates scaffold-free systems optimize scalability for screening while scaffold-based approaches enable physiologically relevant regenerative studies. Integration of both methodologies provides flexibility matching experimental design to scientific objectives, accelerating translation to clinical applications.

Patient-derived organoids (PDOs) have emerged as powerful tools in personalized medicine applicable to both non-malignant conditions and to cancer, where they are increasingly used for personalized drug screening and precision treatment strategies in part due to their ability to replicate tumor heterogeneity. They also serve as study model systems to understand disease mechanisms, pathways, and the impact of ex vivo exposures. We present a detailed step-by-step protocol for generating organoids from normal crypts, polyps, and tumors, including methods for tissue processing, crypt isolation, culture establishment, and the transition from basolateral to apical-out polarity for co-culture and exposure-based studies. The protocol also includes immunofluorescence staining procedures for cellular characterization and quality control measures. Our standardized approach successfully generates organoids from diverse colorectal tissues with high efficiency and reproducibility. This comprehensive guide addresses common technical challenges and provides troubleshooting strategies to improve success rates across different sample types. We believe that this resource will enhance reproducibility in organoid research and expand their utility in translational applications, particularly for personalized medicine approaches in colorectal cancer.

This study aimed to evaluate the placement accuracy and reproducibility of Surface Guided Radiotherapy (SGRT) compared with the conventional tattoo/laser method in patients undergoing radiotherapy for abdominal malignancies. A retrospective analysis was conducted on 43 patients treated with either SGRT (Group A) or the tattoo/laser technique (Group B). Patients in both groups underwent CBCT to quantify the positioning shifts in the vertical (Svrt), lateral (Slat) and longitudinal (Slng) axes, as well as the total shift. Statistical indicators including median, interquartile range (IQR), and range were calculated, and Mann-Whitney U tests were performed due to non-normal data distribution. Median values in all axes were same between groups: Svrt = 0.4 cm, Slat = 0.2 cm, Slng = 0.4 cm. Group A showed a higher total median shift equal to 0.8 cm versus 0.7 cm of Group B. However, IQRs were smaller in the Group B for all directions and total shift, indicating greater method consistency. Statistically significant differences (p < 0.05) were observed in all axes, except the vertical. These findings suggest that, while SGRT achieves comparable median alignment, its use in a highly variable anatomical region such as the abdomen may be associated with greater setup variability.

Maintaining the integrity of cryogenically preserved biological materials is critical, as even brief, undetected temperature excursions in storage can compromise sample viability. Existing monitoring systems may miss transient thaw-refreeze events, posing serious quality risks. To address this, we developed and validated frozen indicator tubes that visually signal deviations from the frozen state, serving as a cost-effective backup to electronic monitors. Our first method uses an aqueous dye solution that immobilizes the dye when frozen; any thawing causes the dye to disperse, providing a clear, external visual cue of a partial or complete thaw. For ultra-low-temperature storage (-80 °C), we introduced a second method using an ethanol-based solution calibrated to indicate thaw events. This system detects temperature rises of 10 °C or more sustained for at least fifteen minutes-conditions that may jeopardize sample stability. When paired with standard monitoring systems, these indicator tubes offer an added layer of protection by providing simple, reliable, and immediate visual confirmation of critical temperature breaches. This innovation enhances confidence in cryogenic storage protocols and supports the long-term preservation of sensitive biological materials.

The bioavailability of a drug is closely linked to its solubility, making its early determination essential in drug development. The saturation shake-flask (SSF) method is the gold standard protocol for this, which includes a phase separation step-either by sedimentation, filtration, or centrifugation. This step is critical, as it can directly influence the accuracy of the results. This study investigated the impact of centrifugation parameters-time and rotation speed-on solubility measurements. Additionally, we compared two sample preparation protocols: continuous stirring for 24 h versus 6 h of stirring followed by 18 h of sedimentation before centrifugation. Four model compounds were tested at three pH values using Britton-Robinson buffers. Centrifugation was conducted for 5, 10, or 20 min at either 5000 or 10,000 rpm. Results showed that pre-sedimented samples yielded solubility values closer to sedimentation-only references, while continuous stirring often led to overestimated values, particularly at higher speeds and longer durations. One such example was papaverine hydrochloride, that showed solubility values 60-70% higher than the reference after centrifugation at 10,000 rpm for 20 min without prior sedimentation. Lower standard deviations were observed with shorter, slower centrifugation, with 5 min and 5000 rpm yielding results closest to the reference values.

Antimicrobial resistance is a global health issue, and the WHO has made significant efforts in the development of tools for its monitoring. However, such tools are underutilized, due to limited knowledge, technical capacity, and scarcity of economic resources. AMR surveillance can be conducted using WHOnet and R, two free-of-charge software tools widely adopted in both clinical practice and scientific research. WHOnet is designed for managing laboratory data and antimicrobial susceptibility test results, while R is a programming language dedicated to statistical computing and data visualization. The combined use of these tools enables a reproducible workflow for retrospective AMR trend analysis. This paper provides step-by-step instructions on how to perform such analysis and also provides the respective R code. The described code and software results are shown using real-world data from an Italian hospital as an example. The standardization of the analysis process and the rapid availability of data on antimicrobial resistance are critical for both clinicians and public health professionals. They would allow for empirical decisions on antimicrobial treatment based on the specific epidemiological characteristics of the hospital or community setting.

The hyperinsulinemic-euglycemic clamp technique is considered the gold standard for measuring insulin sensitivity in large animals. We developed a practical method for conducting concurrent glucose clamp experiments in a pair of sedated farm swine positioned in a sling. Descriptions of customized equipment and central venous access surgical procedures for blood collection are provided. Personnel functions are described for execution of the clamp protocol. A total of 24 hyperinsulinemic-euglycemic clamp studies were performed over 6 weeks. Infusaports remained functional for 1454 blood samples. There were three CSII catheter occlusions during bolus administration, and the swine showed no signs of infection or disease. IM telazol at 1.0 mg/kg, administered 1-2 h prior (mean of 3.26 mL ± 1.59) was effective in keeping animals comfortable. SpO₂ and heart rate remained within normal ranges. Means ± SD total infused volumes for octreotide, 10% dextrose, and saline were 9.7 ± 0.93 mL, 2328.0 ± 672.8 mL, and 690.3 ± 206.8 mL. Mean blood glucose was maintained between 75.7 and 87.8 mg/dL (CV 3.17%) for the 24 experiments. The GIR infusion rate peaked between 15 and 60 min after insulin bolusing, with insulin C_max of 108.5 pmol/L and t_max at 10 min. All aspects of the protocol were effectively carried out. The animals remained in good health, and the implanted infusion ports remained patent for over 700 blood draws per animal. This method could potentially reduce the number of animals used and the costs of other similar experiments.

Major depressive disorder (MDD) is a prevalent and disabling condition. Transcranial direct current stimulation (tDCS) may improve symptoms by modulating neuroplastic and inflammatory mechanisms. This randomized, double-blind, placebo-controlled trial will recruit adult outpatients with MDD showing residual symptoms despite at least four weeks of stable SSRI treatment. Participants will be randomized to active or sham add-on tDCS while continuing their antidepressant regimen. The intervention will consist of 15 sessions over 3 weeks, targeting the left dorsolateral prefrontal cortex (anode F3, cathode F4) at 2 mA for 30 min per session. The primary outcome is the reduction of depressive symptoms measured by the Hamilton Depression Rating Scale-17 (HDRS), with remission defined as HDRS-17 ≤ 7. Secondary outcomes include cognitive performance (attention, executive functioning, memory), serum biomarkers (BDNF, VEGF, NGF, NRG1, angiogenin, IGF1, IL-6, TNF-α), cortical excitability assessed by transcranial magnetic stimulation (motor threshold, silent period, intracortical inhibition/facilitation), and cerebral hemodynamics by transcranial Doppler sonography (blood flow velocity, pulsatility, resistivity). Assessments will occur at baseline, post-treatment, and 3- and 6-month follow-ups. This trial aims to evaluate the efficacy of adjunctive tDCS in MDD with residual symptoms and its biological correlates, bridging clinical improvement with electrophysiological and neurovascular mechanisms.

Dermal tissue samples are a rich source of germplasm because they can be readily collected, frozen as part of a genebank collection, digested and cultured, and used for a variety of purposes such as genotyping or other forms of genetic research. Derived fibroblasts can also be used for somatic cell nuclear transfer, and the remaining cells can be frozen for future use. However, collection of tissues with ear notchers, scalpels, or biopsy punches can be problematic because tissue handling and the tool surfaces can contaminate the samples. Therefore, the modification of the Allflex Tissue Sampling Unit (TSU) system was explored to determine if the technology can empower rapid collection of clean samples that are easily identifiable and portable. Results indicate that the TSU system was efficient, and samples that were collected and processed for tissue culture resulted in successful derivations of fibroblasts from 7 of 11 animals. Thus, the TSU system appears to be a viable option for collecting and preserving dermal tissue for genebanking and other applications where simple, rapid collection of large quantities of samples is required.