Journal of Visualized Experiments

This protocol describes a Langendorff-based method for isolating intact adult mouse ventricular myocytes using syringe pump-driven perfusion. The approach retains the key physiological advantage of the conventional Langendorff technique, continuous retrograde coronary perfusion, while simplifying the overall setup. By combining retrograde aortic perfusion with widely available laboratory equipment, the method provides an accessible alternative to traditional Langendorff systems. A precision syringe pump connected to an in-line heater is used to deliver temperature-controlled, constant-flow perfusion during enzymatic digestion. In contrast to gravity-driven constant-pressure systems, constant-flow perfusion maintains stable enzyme delivery despite changes in coronary resistance that occur during tissue digestion. Use of an inline heater allows precise, rapid temperature-controlled delivery, avoiding the complexity, leak risk, thermal lag, and contamination susceptibility associated with traditional water-jacketed systems. Our setup reduces variability in perfusion rate and minimizes susceptibility to occlusion, flow interruption, or compliance-related artifacts, enhancing reproducibility. The method consistently yields adult ventricular myocytes with high viability (>70% rod-shaped, calcium-tolerant), enabling a broad range of functional analyses including electrophysiology, contractile performance and calcium handling. Step-by-step instructions, troubleshooting guidance, and anticipated outcomes are provided to facilitate adoption in laboratories without dedicated isolated-heart perfusion infrastructure. Key FeaturesO_LISimplified Langendorff-based mouse cardiomyocyte isolation method that eliminates the need for specialized perfusion rigs. C_LIO_LISyringe pump-driven constant-flow perfusion combined with inline temperature control improves reproducibility by ensuring stable enzyme delivery and precise temperature regulation. C_LIO_LIGenerates high-yield, calcium-tolerant adult mouse ventricular myocytes suitable for functional studies. C_LI Graphical Overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/718810v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@15061cdorg.highwire.dtl.DTLVardef@44fd48org.highwire.dtl.DTLVardef@1509285org.highwire.dtl.DTLVardef@c362a4_HPS_FORMAT_FIGEXP M_FIG Graphical overview of the simplified Langendorff-based mouse cardiac myocyte isolation protocol. C_FIG

Optimized histological techniques are crucial for visualizing cellular morphology across zebrafish tissues. Here, we report a rapid and reliable hematoxylin and Oil Red O (H-ORO) staining protocol for frozen sections that can be completed in less than three minutes. Mayers hematoxylin is used for nuclear staining, followed by Oil Red O (ORO) to visualize lipid-rich structures such as the endomysium surrounding myofibers, white matter of the brain, and myelin layers of major axonal tracts. Importantly, our optimized H-ORO protocol preserves tissue integrity and minimizes artifacts such as myofiber shrinkage commonly observed with ethanol-based hematoxylin and eosin (H&E) staining in both frozen and paraffin sections.

BackgroundPlatelet-derived Growth factors play key roles in tissue repair and regeneration, yet conventional platelet-rich plasma (PRP) formulations release these mediators inconsistently in vivo due to variability in platelet yield and activation dynamics. To overcome this limitation, direct administration of concentrated platelet-derived growth factor preparations has gained interest, though current manufacturing approaches for human platelet lysate (hPL), growth factor concentrates (GFC), and conditioned serum remain constrained by batch variability, incomplete platelet degranulation, and reliance on anticoagulants. Here, we examine alternative platelet activation workflows to establish a standardized, efficient, and reproducible method for high-yield growth factor recovery suitable for translational and clinical applications. MethodsNine GFC production protocols were compared, employing different combinations of freeze-thaw (FT) cycling, glass bead (GB) agitation, calcium (Ca2) activation, and a novel Enriched Growth Factor (Enriched-GF) method. The objective was to identify a protocol capable of maximizing growth factor yield within a three-hour workflow. Optimal Ca2 concentrations and GB conditions were determined from prior optimization studies and integrated into the Enriched-GF processing scheme. Platelet concentrates (n = 10 per protocol) were processed under each condition, and growth factor levels were quantified using ELISA. ResultsGrowth factor yields differed significantly across protocols. The greatest and most consistent increases in growth factor release were observed with the Enriched-GF method combining GB activation, FT cycling, and Ca2 stimulation. This approach resulted in markedly elevated concentrations of key regenerative mediators, including enhanced EGF release, a 4.5-fold increase in PDGF, maximal TGF-{beta} liberation, and a four-fold increase in FGF2 relative to conventional platelet lysate or conditioned serum preparations. These results were reproducible across independent donor pools, demonstrating robustness and batch-to-batch consistency. ConclusionWe describe a rapid and reproducible method for producing highly concentrated platelet-derived growth factors using a combined GB-FT-Ca2 activation strategy. The Enriched-GF protocol consistently outperformed existing platelet lysate, conditioned serum, and conventional GFC preparation methods, yielding a standardized product with enhanced growth factor content. This Enriched-GF approach offers a clinically practicable solution for applications in regenerative medicine requiring reliable and high-yield growth factor delivery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/712883v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1f059d9org.highwire.dtl.DTLVardef@9aeffforg.highwire.dtl.DTLVardef@27cd1org.highwire.dtl.DTLVardef@150b7d1_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic overview of platelet concentrate preparation from whole blood and the generation of different platelet lysates and growth factor-enriched serum using freeze-thaw, calcium gluconate, and glass bead activation methods.

Osteocytes play a central role in bone remodeling, mineral metabolism, and skeletal homeostasis, but direct molecular analysis of human osteocytes remains technically challenging because they are embedded within the mineralized bone matrix. Surgically obtained human bone specimens provide valuable material for studying human bone biology; however, surface-associated cells, marrow-derived cells, and adherent soft tissues can confound downstream transcript analysis. Here, we describe a bone fragment-based protocol for preparing surgically obtained human bone specimens for molecular analysis of osteocyte-associated transcripts. The protocol consists of mechanical trimming, mincing into small bone fragments, repeated washing, and five sequential rounds of collagenase digestion to reduce non-osteocytic cellular components associated with the bone surface and marrow spaces. The remaining mineralized bone fragments are then frozen in liquid nitrogen, cryogenically pulverized, and lysed in TRIzol reagent for total RNA extraction. Histological validation using residual maxillary bone specimens showed that sequential collagenase digestion markedly reduced adherent soft tissue and extra-matrix nuclei while preserving osteocyte lacunar occupancy. This protocol provides a practical workflow for bone fragment-based RNA analysis focused on osteocyte-associated transcripts in human bone specimens. Specifications table O_TBL View this table: org.highwire.dtl.DTLVardef@1cec618org.highwire.dtl.DTLVardef@2f746forg.highwire.dtl.DTLVardef@1854247org.highwire.dtl.DTLVardef@1c26c1aorg.highwire.dtl.DTLVardef@1473a88_HPS_FORMAT_FIGEXP M_TBL C_TBL

Octopus vulgaris and other cephalopods are of increasing interest as neurobiological model organisms. This protocol describes a method to record calcium activity from individual cells in acute brain slices from Octopus vulgaris hatchlings during exogenous application of neurotransmitters. Using this protocol, we characterized single-cell responses to specific neurotransmitters in the optic lobes, which process visual information. The approach is readily adaptable to other cephalopods and small invertebrate species. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=146 HEIGHT=200 SRC="FIGDIR/small/711860v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1564eaeorg.highwire.dtl.DTLVardef@147b682org.highwire.dtl.DTLVardef@11f3b85org.highwire.dtl.DTLVardef@17c9d70_HPS_FORMAT_FIGEXP M_FIG C_FIG

Investigating the neurophysiology of nociception is aided by electrophysiological recording from dorsal root ganglion (DRG) neurons. Because DRG neurons are heterogeneous with overlapping electrophysiological properties, methods to distinguish neuron subtypes are valuable for properly interpreting the measurements and drawing conclusions. Automated patch clamp recording offers an approach for conducting these experiments at higher throughput than conventional recording methods, but identification of neuron subtypes is challenging. We developed a method for recording from acutely isolated mouse DRG neurons using automated patch clamp recording coupled to optogenetic stimulation that was capable of discerning NaV1.8 and TRPV1 expressing neuron subpopulations. This approach can facilitate physiological and pharmacological studies of DRG neurons with potential value in developing and testing targeted analgesic agents.

CaMKII promoter is widely used to label and manipulate hippocampal pyramidal neurons via transgenic mouse lines or viral approaches. While it targets most excitatory neurons, a small subset remains unlabeled and often overlooked. We present an AAV-based strategy combined with CaMKII-driven Cre expression to access and study this remaining population. Furthermore, we provide a detailed protocol for in-house AAV production, targeted stereotaxic delivery, and functional validation of targeted neurons through slice electrophysiology and behavior. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/723440v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@3a31ccorg.highwire.dtl.DTLVardef@9b7e90org.highwire.dtl.DTLVardef@92297borg.highwire.dtl.DTLVardef@1e159eb_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mars relatively moderate surface conditions, availability of solar energy, and in situ resources like water ice, carbon dioxide, and mineral-rich regolith make it a compelling target for supporting life beyond Earth. However, existing experiments testing habitability in Mars conditions generally rely on leachates of physical regolith simulants, which vary in composition across simulant types, leaching conditions, and production batches. We introduce a defined Mars media (DMM) that accurately simulates the biologically relevant nutrients (nitrogen, phosphorus, and sulfur) and stressors (perchlorates, heavy metals) in Martian regolith when it is leached in water at neutral pH. We formulated DMM by combining direct rover and lander measurements from Mars with laboratory measurements of regolith simulant leachates. We validate DMM from a lx to 20x concentrate, equivalent to 40 g/L to 800 g/L of leached regolith. Using DMM with acetate as a Mars atmosphere-derived carbon source, we grew eight heterotrophic bacteria, confirming that organisms can source all essential nutrients from Martian resources. We also show that microbial growth in DMM is robust to uncertainties in Martian regolith composition: sensitivity experiments can identify limiting trace element nutrients and toxins in DMM, and demonstrate that bacterial growth is maintained across at least an order of magnitude variation in their concentrations. This is the first defined Mars regolith media recipe containing both macro- and micro- nutrients, and designed specifically for biological experimentation. By shifting from variable leachate-based approaches to a defined aqueous analog, we enable controlled hypothesis testing of microbial survival, growth, and function. DMM will enable further research on astrobiology, biological in situ resource utilization, large-scale soil remediation, and terraforming. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/719001v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1314b20org.highwire.dtl.DTLVardef@13b57d4org.highwire.dtl.DTLVardef@103315eorg.highwire.dtl.DTLVardef@9e18fe_HPS_FORMAT_FIGEXP M_FIG C_FIG

Transcriptional bursts regulate gene expression by altering burst size or burst frequency. Here, we present a protocol that integrates fixed-cell smFISH and live-cell single-molecule imaging to analyze estrogen-responsive transcriptional bursting of the TFF1 gene in human breast cancer cell lines. This workflow enables measurement of burst size, burst initiation, and active allele frequency to determine how endocrine disruptor chemicals modulate transcriptional bursting dynamics. For complete details on the use and execution of this protocol, please refer to Day, Yasar et al.1

As human space exploration expands to the Moon, Mars, and beyond, there is a growing need to study the effects of altered gravity on the microbial systems that we will bring with us for life support. Because spaceflight experiment opportunities are rare and resource-intensive, most space biology experiments are conducted using ground-based simulators. The most common microgravity simulator for microbial experiments, the rotating wall vessel, can approximate the low-shear and low-turbulence conditions that characterize microgravity. However, current designs do not allow for real-time measurement of growth or metabolic activity during rotation: experiments require destructive sampling or disruption of the microgravity simulation conditions. Here, we describe the development of an in situ spectroscopy system compatible with the Cell Spinpod rotating wall vessel, which enables measurement of both optical absorbance and fluorescence with high temporal resolution, producing growth curves similar to those from an off-the-shelf plate reader. These results are validated using two common microbial hosts: Escherichia coli and Saccharomyces cerevisiae. The Spinpod Optical System has the potential to diversify the types of microbiology experiments possible in simulated microgravity, allowing the measurement of not only growth curve parameters but also metabolic activity, gene expression, or community dynamics. It thus has the potential to improve the quality of experiments seeking to characterize microbial responses to spaceflight conditions.

IntroductionIntraoperative glycemic dysregulation, including unrecognized hypoglycemia and stress-induced hyperglycemia, is common during elective surgery. Conventional point-of-care (POC) monitoring provides only intermittent measurements, limiting the anesthesiologists ability to detect rapid glucose fluctuations. Continuous glucose monitoring (CGM) enables real-time, trend-based assessment, potentially shifting intraoperative glycemic management from reactive to proactive. ObjectiveTo meta-analyze the analytical accuracy, intraoperative glycemic efficacy, and feasibility of subcutaneous CGM in adults undergoing elective surgery, informing anesthesiology practice. MethodsThis systematic review and meta-analysis followed the PRISMA 2020 statement. Searches were conducted in PubMed, Embase, and Cochrane Central Register of Controlled Trials from January 2010 to May 2025. Eligible studies included randomized controlled trials and prospective cohorts of adults undergoing elective surgery under general or neuraxial anesthesia using subcutaneous CGM. Primary outcomes were pooled mean absolute relative difference (MARD) and time in range (TIR, 70-180 mg/dL). Random-effects models were applied. ResultsTen studies (3 RCTs, 7 cohorts; N=557) were included. Pooled MARD was 14.1% (95% CI 11.3-16.9%; I{superscript 2}=78%), lower in non-cardiac surgery (12.7%) than cardiac procedures with hypothermia (19.2%; p=0.03). CGM improved TIR by +14.9 percentage points (95% CI 7.2-22.6; p<0.001). Clinically significant hypoglycemia was detected in 43% of patients, all missed by POC. Sensor availability exceeded 96%, with no serious device-related events. ConclusionSubcutaneous CGM provides acceptable intraoperative accuracy and improves glycemic control, supporting its integration into anesthetic management.

BackgroundPhotobiomodulation (PBM) therapy has demonstrated therapeutic potential in promoting cellular repair, modulating inflammation, and enhancing mitochondrial function. Platelet-rich plasma (PRP) is widely used in regenerative medicine due to its concentration of growth factors and cytokines. Very small embryonic-like stem cells (VSELs), a rare population of pluripotent stem cells present in adult tissues, have emerged as a potential contributor to tissue regeneration. While PBM and PRP are used in combination, how VSELs or Multi-lineage stress enduring (MUSE) cells are at play, and the biological mechanisms underlying their synergistic effects remain incompletely characterized. ObjectiveThis exploratory pilot study aimed to evaluate whether application of the MD Biophysics laser to autologous PRP is associated with measurable changes in VSEL-related antibody marker expression, and to identify directional trends to inform future controlled studies. MethodsPRP samples were collected from participants across seven test dates (July 2024 to February 2025), yielding 18 participant-session datasets. Samples were analyzed before (Pre) and after (Post) laser application using flow cytometry conducted at a UCLA Flow Cytometry Laboratory. Four VSEL-associated antibody markers were assessed: CD45-CD34+, CXCR4+, CD133+, and SSEA-4+. Analyses were descriptive and focused on paired differences and directional trends due to the exploratory design and absence of a control group. ResultsThree of four VSEL-associated markers (CXCR4+, CD133+, and SSEA-4+) demonstrated a group-level increase in median paired differences following laser application. Directional increases were observed in 12/18 sessions for CXCR4+, 10/18 for CD133+, and 9/18 for SSEA-4+. CD45-CD34+ showed a near-equal distribution of increases and decreases. Ki-67 positivity indicated the presence of viable, proliferative cells. While no findings reached statistical significance due to limited sample size, consistent directional trends were observed across multiple markers. ConclusionApplication of PBM to autologous PRP was associated with directional increases in multiple VSEL-associated antibody markers, suggesting a potential role for stem cell activation or mobilization in the mechanism of action. Although preliminary and not statistically powered, these findings provide hypothesis-generating evidence supporting further investigation. The observed trends informed iterative protocol refinement and establish a foundation for future controlled, adequately powered studies to evaluate clinical efficacy and underlying biological mechanisms.

Genetically encoded biosensors are GFP-based tools that can visualize the dynamics and spatial features of cellular processes. The design of a genetically encoded biosensor dictates the method that is used to measure the response. Common read-outs use some sort of fluorescence intensity measurement, which is subject to both technical and biological perturbations, including sample drift, excitation power fluctuations, changes in sample size/volume, or a change in expression level. Yet, the fluorescence lifetime of a fluorophore is not affected by the aforementioned perturbations. Therefore, biosensors that respond with a large lifetime change offer a more robust method of detecting cellular processes. Here, we report on protocols for calcium imaging using fluorescence lifetime imaging microscopy (FLIM) to measure the response of a genetically encoded lifetime biosensor. The protocols include details on biosensor production and purification, calibration of purified biosensor with FLIM, introduction of the plasmid in HeLa and endothelial cells, and timelapse analysis of FLIM data. In this chapter we use the green fluorescent biosensor G-Ca-FLITS as an example but the protocols can be generally applied to biosensors with lifetime contrast. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/717680v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@167f612org.highwire.dtl.DTLVardef@4c5603org.highwire.dtl.DTLVardef@1a2eb6borg.highwire.dtl.DTLVardef@10ddc63_HPS_FORMAT_FIGEXP M_FIG C_FIG

AimsThe development of a method for isolating mitochondria from a specific cell type within a given tissue, while preserving their structural and functional integrity to the greatest possible extent, remains an ongoing challenge. The aim of this study was to establish a protocol for the isolation of mitochondria from rodent cardiomyocytes, characterized by minimal contamination with other cell types and a high yield of mitochondrial fractions originating from distinct subcellular regions of cardiomyocytes. Methods and resultsIn the present study, cardiomyocytes from guinea pig and rat hearts were isolated using a standard enzymatic digestion protocol in a Langendorff heart perfusion system. Traditionally, the isolation of organelles, including mitochondria, from whole cardiac tissue as well as from cardiomyocytes has relied primarily on mechanical tissue homogenization These conventional approaches involve the localized application of high pressure to cells, which may potentially damage delicate organelles, particularly mitochondria. Moreover, such homogenization preferentially releases mitochondria located in the subsarcolemmal region of cardiomyocytes rather than representing the entire mitochondrial population. In our study, we employed an alternative approach based on the gentle mechanical disruption of cardiomyocytes by passing the cell suspension through selected cell strainers using a cell scraper. This strategy facilitated mild disruption of cellular structures, significantly increasing the yield of mitochondria released from interfibrillar regions while preserving mitochondrial functionality. Moreover, this method decrease probability of sample contamination with mitochondria from other cells, based on cell size differences. The effectiveness of this method was confirmed by transmission electron microscopy, and high-resolution respirometry, which revealed no evidence of outer mitochondrial membrane damage, as indicated by the lack of response to the addition of exogenous cytochrome c to the incubation chamber. Moreover, mitochondrial oxygen consumption increased by 7.39 {+/-} 1.25-fold following the addition of 100 {micro}M ADP, reflecting efficient ADP-stimulated respiration. Furthermore, fluorescence measurements were performed. to assess changes in the mitochondrial inner membrane potential ({Delta}{Psi}). The isolated mitochondria were also suitable for electrophysiological studies using the single-channel patch-clamp technique. Additionally, mitochondria isolated using the protocol developed in our laboratory exhibited a high capacity for transplantation into H9c2 cells. ConclusionIn summary, our mitochondrial isolation method is rapid, efficient, and yields functionally competent mitochondria. These preparations are suitable for a wide range of downstream applications, including patch-clamp electrophysiology, analyses of oxygen consumption under various pharmacological conditions, as well as mitochondrial transplantation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=162 HEIGHT=200 SRC="FIGDIR/small/716092v1_ufig1.gif" ALT="Figure 1"> View larger version (85K): org.highwire.dtl.DTLVardef@613495org.highwire.dtl.DTLVardef@1c34338org.highwire.dtl.DTLVardef@722900org.highwire.dtl.DTLVardef@e1f7a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

1Sex steroid hormones are not exclusively localised in the circulation and can be found in numerous extragonadal tissues, in concentrations unrelated to the circulating fraction. Existing methodology to measure intramuscular steroid hormone concentrations includes both immune-based assays and liquid chromatography-mass spectrometry (LC-MS), the gold standard for hormone measurements. To date, no LC-MS based methods validation has been published on the measurement of intramuscular sex steroid hormones, despite clear biological relevance. Here, we describe the development and validation of a simple, high-throughput LC-MS Orbitrap method for the measurement of 10 intramuscular sex steroid hormones, including pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, epitestosterone, dihydrotestosterone, oestrone, oestradiol, and oestriol. In brief, isotope labelled standards were added to 5-6 milligrams of lyophilised muscle tissue, homogenised and extracted with ethyl acetate. The extracts were dried down and sequentially derivatised with 1-methylimidazole-2-sulfonyl chloride and hydroxylamine hydrochloride to target both the phenolic hydroxyl groups and ketone groups. The limit of detection was 1.0 {+/-} 1.0 pg/mg (range 0.36 - 3.26 pg/mg), with a R2 > 0.99 for all analytes. Matrix effects were 90-110% for all analytes except for dihydrotestosterone (143.6%), and precision was <10 CV% for all analytes in the presence of a muscle matrix. Our method allows for 20-40 samples to be prepared in [~]4 h, with a sample data acquisition time of 13 minutes. Moreover, our method provides the opportunity for specific analysis of steroid hormone concentrations in skeletal muscle, allowing target tissue specificity instead of relying on proxy measures from the circulation.

Coenzyme A is an essential cofactor synthesized from pantothenate, cysteine, and ATP, and is involved in numerous processes of cellular metabolism through its ability to carry activated acyl groups. Coenzyme A participates in catabolism of carbohydrate, fat and amino acids; biosynthesis of fatty acids, cholesterol and heme; and protein modification including acetylation and 4-phosphopantetheinylation. Despite CoAs critical functions, the regulation of CoA levels and the rate of CoA synthesis in different cell types and disease states are not well understood. One reason for this gap is that many acyl-CoA species are analytically challenging to measure due to factors including instability, poor ionization, and the wide range of biochemical properties conferred by different acyl chain lengths. In addition, most current methods do not support analysis of CoA isotopic labeling, which is required to quantify CoA synthesis rate or to measure absolute concentration using isotope-labeled internal standards. Here, we describe a method to quantify the concentration and isotopic labeling of total CoA, defined as the sum of CoASH plus all acyl-CoA species. Acyl-CoA species are hydrolyzed using sodium hydroxide to remove acyl chains, then CoA is derivatized on the thiol with N-ethylmaleimide (NEM). Following protein precipitation and solid phase extraction, samples are analyzed by liquid chromatography-mass spectrometry. This method is linear in a wide range that captures mouse tissue CoA levels, with accuracy within 15% error and precision below 15% relative standard deviation for both pure standards and tissue samples. We applied this method to measure total CoA concentration in five tissues from male and female mice, and total CoA synthesis rate in mouse liver via infusion of 13C-15N-pantothenate. Overall, this method offers a tractable approach to measure total CoA concentration and isotopic labeling to enable study of total CoA synthesis rates and concentrations in health and disease.

Computational bioacoustics has seen significant advances in recent decades. However, the rate of insights from automated analysis of bioacoustic audio lags behind our rate of collecting the data - due to key capacity constraints in data annotation and bioacoustic algorithm development. Gaps in analysis methodology persist: not because they are intractable, but because of resource limitations in the bioacoustics community. To bridge these gaps, we advocate the open science method of data challenges, structured as public contests. We conducted a bioacoustics data challenge named BioDCASE, within the format of an existing event (DCASE). In this work we report on the procedures needed to select and then conduct useful bioacoustics data challenges. We consider aspects of task design such as dataset curation, annotation, and evaluation metrics. We report the three tasks included in BioDCASE 2025 and the resulting progress made. Based on this we make recommendations for open community initiatives in computational bioacoustics.

AbstractsThe Ploton silver method employs a 50% silver nitrate solution (w/v; 2.943 mol/L) for staining and quantitative analysis of the osteocyte lacuno-canalicular system (LCS). We previously demonstrated that lower silver nitrate concentrations (0.5-1 mol/L) stain the LCS more effectively, revealing a greater number of LCS than the Ploton silver method. However, the staining duration of our initial modified method (60 minutes) remained comparable to that of the Ploton silver method (55 minutes), limiting its broader adoption. Here, we developed a rapid silver nitrate staining method by systematically evaluating the effects of temperature on staining efficacy. We found that incubation at 50-70{degrees}C for 10 minutes with a 1 mol/L silver nitrate solution produced optimal results. This rapid high-temperature method achieved excellent LCS visualization in bone samples from multiple animal species and in mouse pathological models. Moreover, high-temperature staining mitigated the LCS damage and insufficient staining associated with the 50% silver nitrate solution used in the Ploton silver method. This rapid 10-minute silver staining technique, designated the Wu-Wang silver method, provides a more accurate and efficient approach for LCS staining and quantitative analysis. Its adoption will facilitate systematic characterization of LCS morphological variations across vertebrate species, thereby advancing our understanding of osteocyte morphogenesis and the pathogenic mechanisms underlying bone and joint diseases. Graphical abstract (Created in BioRender, https://BioRender.com) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/719546v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@996f66org.highwire.dtl.DTLVardef@160b1a5org.highwire.dtl.DTLVardef@12eee4corg.highwire.dtl.DTLVardef@1edce8_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIElevating the staining temperature to 50-70{degrees}C enabled rapid and efficient silver nitrate staining of the osteocyte lacuna-canalicular system (LCS) within 5-10 minutes using 1 mol/L silver nitrate. C_LIO_LIThe high-temperature Wu-Wang silver method outperformed the conventional Ploton silver method, providing superior osteocyte LCS visualization while eliminating issues of osteocyte LCS damage and insufficient staining observed with the Ploton silver method. C_LI

In vitro cytotoxicity assessments frequently rely on staining-based methods that indirectly estimate viable cell numbers indirectly. A major limitation of many such techniques is their endpoint nature, requiring cell lysis or irreversible processing that precludes longitudinal monitoring of cellular responses following treatment. An ideal assay for evaluating cell viability and proliferation should be simple, rapid, cost-effective, reproducible, and highly sensitive, while also enabling accurate quantification with minimal interference from test compounds. The resazurin reduction assay satisfies these criteria, offering a sensitive and economical alternative to conventional tetrazolium-based methods. Although both assay types depend on the metabolic reduction of a dye by viable cells, they differ mechanistically. Tetrazolium salts (e.g., MTT) are reduced by cellular dehydrogenases to insoluble formazan crystals that require solubilization before to detection. In contrast, resazurin--a cell-permeable, non-fluorescent blue dye--is reduced to resorufin, a highly fluorescent compound detectable without additional processing steps. This property renders the resazurin assay broadly applicable to viability testing in eukaryotic cells cultured in both 2D and 3D formats, as well as in bacterial systems. Here, we present a streamlined, universal protocol for implementing the resazurin reduction assay across diverse experimental models, emphasizing its practicality, reproducibility, and adaptability for real-time viability monitoring. Key featuresO_LIReal-time, non-destructive monitoring: Enables longitudinal studies by allowing repeated measurements of the same samples over hours without toxicity or disruption. C_LIO_LIStreamlined workflow: A simple "add-incubate-read" protocol eliminates the need for cell lysis, washing, or extraction, saving time and reducing variability. C_LIO_LIBroad sample compatibility: Versatile and reliable for use with 2D monolayers, 3D spheroids, organoids, and bacterial cultures. C_LIO_LIHigh sensitivity: Fluorescent detection of resorufin provides exceptional sensitivity, enabling accurate quantification of even small viable cell populations. C_LIO_LILow background and minimal interference: A clean fluorescent readout reduces the risk of signal artifacts, offering a more reliable alternative to traditional colorimetric assays. C_LIO_LICost-effective and accessible: Utilizes standard laboratory plate readers and commercially available reagents, making it an economical choice for any lab. C_LIO_LIScalable for high-throughput screening: Easily adaptable to various plate formats, supporting both small-scale experiments and large-scale automated screening applications. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/718248v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@82bcecorg.highwire.dtl.DTLVardef@14164aforg.highwire.dtl.DTLVardef@395118org.highwire.dtl.DTLVardef@fb1349_HPS_FORMAT_FIGEXP M_FIG C_FIG

Dance is a core form of human-environment interaction and a powerful medium for emotional expression, yet dancers are routinely exposed to environmental affective cues that may shape their movement. We tested whether a negative emotional context induced immediately before improvisation alters dance biomechanics. Twenty professional dancers performed two 3-min improvised dances. Between dances, they viewed either Neutral or Negatively valenced pictures from the International Affective Picture System (IAPS; 2 min 40 s, 5 s per image). Eye tracking verified attention to the visual stream. Mood was assessed at four time points (PT1-PT4) using the Brazilian Mood Scale (BRAMS), and full-body, three-dimensional kinematics were captured at 300 Hz using a 9-camera optoelectronic system (Qualisys) and processed to measure global movement amplitude and expansion. Negative IAPS exposure increased tension, depression, fatigue, and decreased vigor from PT2 to PT3. Biomechanically, the Negative Stimulus dancers showed a significant reduction in global movement amplitude after negative IAPS exposure, with reduced movement amplitude of the body extremities. In contrast, global movement expansion remained unchanged; that is, the extremities were not positioned closer or farther from the pelvis. Neutral images produced no mood change and no measurable modulation of movement amplitude or expansion. Together, these results support the hypothesis that improvised dance carries biomechanical signatures of the dancers current affective state, beyond the intended expressive content, and provide an automated motion-capture workflow for studying emotion-movement coupling in spontaneous dance. HighlightsNegative visual context shifted dancers mood toward negative affect Negative images reduced movement amplitude in improvised dance Movement expansion remained stable despite mood induction Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/711707v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@aeaacdorg.highwire.dtl.DTLVardef@14f9bf5org.highwire.dtl.DTLVardef@18805fcorg.highwire.dtl.DTLVardef@1411256_HPS_FORMAT_FIGEXP M_FIG C_FIG