BioTechniques

Context: Cytological specimens allow minimally invasive, repeated sampling for diagnosis and molecular testing. However, cytology biobanking protocols have primarily been optimized for morphology, and the effects of preanalytical handling and storage on intracellular proteins remain unclear. Objective: To assess how fixation, staining, storage temperature, and duration affect intracellular protein preservation in cytological specimens and to identify conditions suitable for in situ proximity ligation assay (isPLA). Design: Human cancer cell lines were processed under different preanalytical conditions. Intracellular proteins and protein protein interactions were assessed by isPLA, fluorescence microscopy, and quantitative image analysis. Slides were stored at room temperature or minus 20 Celsius, with or without MayGrunwaldGiemsa (MGG) staining. Results: Storage temperature had the greatest effect on protein signal. Unstained slides stored at minus 20 Celsius retained strong signals for up to 24 months, whereas room temperature storage reduced detectable protein interactions by approximately 95%. MGG staining further reduced signal intensity during prolonged storage. Nevertheless, stained slides remained suitable for isPLA when staining, microscopic evaluation, destaining, and antibody labeling were completed on the same day. Prolonged room temperature storage of stained slides caused loss of antigenicity, increased autofluorescence, and false negative findings. Conclusions: Low temperature storage and standardized preanalytical handling preserve intracellular protein signals in cytological specimens. These conditions support reliable single cell protein profiling of archived samples and their use in biomarker research and translational pathology.

Motivation: Real-time qPCR data are commonly analyzed to obtain a Cq values, which are used to compare the relative abundance of a targeted template. However, improper background subtraction distorts Cq and changing the assigned threshold can alter experimental conclusions. In addition, a Cq values provide no information on the quality of the qPCR for a given sample. Alternatively, global fitting of qPCR data provides agnostic quantification without the use of Cq or thresholds, and the fitting terms provide a useful metric of reaction performance. Current implementations of global fitting are limited to custom in-house programs. Results: qPyCR is comprehensive analysis tool written in Python that performs background signal subtractions, computes a threshold, and reports Cq values in a traditional manner. The program also performs global fitting and reports template abundances along with reaction quality values. The quality values can be used to assign performance standards, which allow researchers to distinguish aberrant reactions from true changes in template abundance. Availability and Implementation: Code and example data are freely available on GitHub (https://github.com/sdmoore-labs/qPyCR) and on Zenodo (https://doi.org/10.5281/zenodo.21115342).

Environmental DNA (eDNA) has emerged as a transformative tool for biodiversity monitoring, offering a non-invasive means to detect and identify species across various ecosystems. However, despite its potential, current eDNA methodologies have significant limitations, including limited sensitivity, restriction to a specific taxonomic group, lengthy processing times, the need for specialized laboratory conditions and equipment, as well as computational bottlenecks in equipment, connectivity, and algorithms. Overcoming these limitations is crucial for the real-time monitoring of a high number of taxa in field-based scenarios. Here we present an integrated eDNA workflow from extraction to data analysis that mitigates several of these challenges through several technical innovations. Our approach adapts the Tagsteady protocol to Nanopore sequencing, integrates multiplex PCR for simultaneous plant and animal detection, and introduces RTBioScan, a real-time analysis pipeline employing hierarchical basecalling and OTU-level consensus generation to achieve high-accuracy taxonomic assignments during sequencing. Using a single set of universal primers, we integrate metabarcoding and barcoding into a cohesive protocol, utilizing universal markers for both animals and plants (cytochrome oxidase 1 region, COI, and Internal Transcribed Spacer 2, ITS2). This comprehensive approach can detect and identify a broad spectrum of species, from individual organisms to entire communities in a single workflow.

BACKGROUND: The protein 4.1a/4.1b ratio is an intrinsic molecular marker of mean red blood cell (RBC) age resulting from progressive deamidation of protein 4.1b during RBC circulation. Despite its potential for estimating mean RBC age from a single blood sample, its application has been limited by labor-intensive RBC membrane preparation, and its interindividual variability has not been characterized in a large human population. METHODS: We developed a low-volume method for direct RBC membrane isolation from whole blood using saponin permeabilization and differential centrifugation without prior RBC washing. Membrane proteins were separated by SDS-PAGE, and protein 4.1a/4.1b ratios were determined by densitometric analysis with Gaussian peak fitting. Assay conditions, sample-volume requirements, membrane protein recovery, and analytical reproducibility were evaluated. The method was subsequently applied to 114 anonymized residual samples from routine diagnostic testing selected for complete blood count parameters within the corresponding laboratory reference intervals. RESULTS: The optimized procedure used 50 uL of whole blood, 0.06% saponin, and centrifugation at 9000 g, although protein 4.1a/4.1b remained measurable from starting volumes as low as 5 uL. Mean within-subject CVs were 3.90% for electrophoretic/densitometric determination and 5.39% for membrane protein recovery; whole-procedure CVs in 3 subjects were 1.40% to 3.50%. In the population analysis, the mean protein 4.1a/4.1b ratio was 1.428 (SD 0.146), with an observed range of 1.052 to1.799 and a central 95% interval of 1.151 to 1.695. Ratios did not differ significantly between females and males (P=0.246) or among females aged 18 to 50 years, females aged >50 years, and males (P=0.151). The ratio showed modest positive correlations with chronological age in females (Spearman p=0.26, P=0.047) and males (p=0.33, P=0.015), while substantial interindividual variability remained across the age range. CONCLUSIONS: Direct isolation of RBC membranes from small volumes of whole blood enables reproducible determination of the protein 4.1a/4.1b ratio using a simplified workflow. Application of the assay to a comparatively large human population revealed substantial interindividual variation in this molecular marker of mean RBC age that was not explained by sex or chronological age alone. The method provides a practical approach for investigating biological variation in mean RBC age and its potential contribution to laboratory measurements influenced by RBC survival.

Marmosets (MAR, Callithrix jacchus) are nonhuman primates extensively studied in a broad array of preclinical biomedical research areas. However, the limited number of available immunological reagents remains a critical shortcoming of these models. We successfully developed monoclonal antibodies (mAbs) pairs that can be used in immunoassays for the identification and quantification of the biomarkers of inflammation C-reactive protein (CRP), CXCL-10 (IP-10), interleukin (IL)-4, IL-6, and IL-10. Recombinant MAR protein variants were produced in mammalian cells, purified and used for mice immunization. Hybridoma clones were selected first for their capacity to bind the recombinant proteins. All possible pair combinations of these mAbs were then tested for their capacity to identify their targets in MAR-derived samples. These MAR samples consisted of supernatant of PBMCs exposed ex vivo to different stimulants, and plasma samples from animals stimulated in vivo with a low-dose intravenous LPS inoculation. The majority of the mAbs that recognized the recombinant proteins failed to bind to the MAR-derived homologs. However, we found MAR-derived reactive mAbs and selected pairs with optimal sensitivity and signal-to-noise ratio for immunoassays that included Luminex multiplexing assays, ELISA, and ELISpot assays. These immunoreagents and assays will improve the translational value of different MAR biomedical models.

Multiple external factors shape human microbial communities. Beverages represent particularly frequent dietary exposures that may impact oral microbiome composition. We examined associations of coffee, tea, alcohol, sugar-sweetened beverages, artificially-sweetened beverages, and fruit juice consumption with the oral microbiome in three large cohort studies (n=5,598). Beverage consumption was assessed by food frequency questionnaire and categorized as non-consumers and tertiles of intake among consumers, except fruit juice, which was categorized into quartiles. DNA from oral wash specimens was sequenced (16S rRNA, V4 region). Associations with alpha and beta diversity and individual genera were assessed using multivariable linear, logistic, count-based regression models and weighted permutation-based models accounting for multiple comparisons in genus-level analyses. Only alcoholic beverage consumption was associated with all alpha diversity metrics; the highest tertile of alcohol drinking was associated with 10.18 (95% CI: 5.56, 14.80) greater observed amplicon sequence variants vs. non-consumers. All beverages accounted for 4.06% of variability in beta diversity (unweighted UniFrac); in univariable models, alcoholic beverages had the largest R2 among individual beverages (0.86%) and accounted for the majority (25/28, 89.3%) of genus-level associations, mostly with beer and liquor. For example, high alcoholic beverage intake (T3 vs. non-consumers) was associated with 2.25 and 2.17 times the odds of having Megasphaera and Peptostreptococcus, respectively, and 23% lower abundance of Lautropia. Alcoholic beverage consumption was strongly associated with oral microbes, including taxa linked to poor oral health; these findings suggest that alcohol-associated differences in the oral microbiome may have implications for oral and systemic health.

Increasing evidence highlights the important role the oral ecosystem plays in systemic health. Despite the fact that oral diseases affect nearly 3.5 billion people globally, our understanding of the precise drivers of these diseases remains limited. To bridge this gap and provide broader understanding of oral public health, a clinical study involving 595 participants 20-78 years of age was conducted over 8 weeks. Characterization of the oral ecosystem using metabolomics, metagenomics, and targeted immune markers were analyzed in conjunction with demographic, lifestyle, and medical history metadata. This comprehensive approach allowed for the identification of specific links between microbial and metabolic signatures and overall wellness, providing a contemporary baseline to enable the development of personalized prevention and treatment strategies to help safeguard oral and systemic public health.

At-home sampling can address obstacles to participation in transcriptomics research. We previously developed the homeRNA and homeRNAmax platforms; these custom-designed tubes containing RNA stabilizer interface with a blood tube compatible with upper-arm blood collection devices, allowing for blood collection and RNA stabilization to occur from a participant's home. In this work we introduce homeRNAdrop, an insert for a commercially available blood tube (BD Microtainer) that divides the tube into two compartments: the top compartment holds 200 L blood and the bottom compartment holds ~590 L liquid stabilizer (RNAlater). By combining blood collection and RNA stabilization into the same tube, homeRNAdrop streamlines sample preparation for participants and allows for more compact sample storage for the lab. In a remote pilot study, n=25 participants from across the United States tested the homeRNAdrop kit. The n=23 samples successfully received by the lab show that samples collected using homeRNAdrop are of sufficient RNA quality (all RINs > 6, a common cutoff for analysis) and yield (mean yield=1.44 g) for downstream gene expression analysis. Almost all participants who successfully collected blood using the Tasso Mini device found the homeRNAdrop device easy to use (n=23/24). Overall, this work adds a simplified, user-friendly platform to the growing suite of tools for remote blood RNA stabilization.

Numerous CRISPR-Cas systems have been developed for molecular detection of genetic elements exploiting the trans-cleavage activity of LbCas12a combined with short fluorescent probes. Alongside, a large variability of buffer conditions has been reported. However, how solution chemistry balances enzymatic turnover, signal stability, and detection performance have not been systematically defined. Here, we evaluated how the interplay between anions (chloride vs. acetate), additives, sequence probes, and ionic strength contributes to LbCas12a trans-cleavage activity. Buffer composition screening revealed that chloride-based buffers maintain a low background but slow catalytic rates, meanwhile acetate-based buffers accelerate enzymatic turnover with and without the target DNA sequence, increasing non-specific signals. Reducing agents and surfactants were the best enhancer combinations, improving cleavage performance by increasing ternary complex formation (Km). High salt concentrations only slightly improved the reaction kinetics. Finally, the 5FAM-TTATT-3BHQ-1 reporter showed the best balance between signal intensity and nonspecific background. Our findings indicate that LbCas12a tolerates a vast buffer composition landscape, maintaining robust trans-cleavage activity and detection performance. However, conditions that enhance catalytic activity simultaneously increase nonspecific probe cleavage and higher background, resulting in lower detection performance. Therefore, assay optimization should prioritize detection performance rather than maximal fluorescence output, where solution chemistry is a critical determinant.

Introduction: Thyroid diseases are major health problems and among the most common endocrine disorders worldwide, including in Ethiopia. They are characterized by alterations in hormone secretion, enlargement of the thyroid gland, or both. Fine needle aspiration cytology is an important initial screening tool, and histopathologic examination is essential for the definitive diagnosis of thyroid disease. This study aimed to compare the diagnostic accuracy of fine needle aspiration cytology with histopathology in identifying thyroid diseases. Methods: A cross-sectional study was conducted at three health facilities in Hawassa City, Ethiopia, involving 304 patients who underwent both thyroid fine needle aspiration cytology and histopathologic examination of the same site between 30th October 2024 and 29th October 2025. FNAC results were classified using the Bethesda system, with histopathology as the gold standard. Data were entered into Kobo Toolbox, exported to SPSS version 27, and analyzed using cross-tabulation and chi-square ({chi}{superscript 2}) tests to determine diagnostic accuracy. Result: 304 patient records were included in the study. The fine needle aspiration cytology was determinate/diagnostic in 81.6% of cases. The study revealed the sensitivity and specificity of fine needle aspiration cytology to be 88.2% (CI 77.6-95.1%) and 94.4% (CI 90.6-97.1%), respectively. The false negative and false positive rates in this study were 2.4% and 4.4%, respectively. The positive predictive value was 80.3% (CI 68.7-89.3%), and the negative predictive value was 96.8% (CI 93.8-98.7%). The accuracy of fine needle aspiration cytology in differentiating benign from malignant thyroid lesions was 93.1%. Conclusion: FNAC demonstrated strong agreement with histopathology and remains a reliable, cost-effective first-line diagnostic tool for thyroid disease, particularly in resource-limited settings. Strengthening sample handling, ultrasound guidance, and adherence to Bethesda criteria may further improve diagnostic precision

While different antigen-presenting cell (APC) populations can drive divergent T-cell fates even when presenting identical antigens (Ags), our ability to trace these specific in vivo interaction histories remains constrained by a lack of compatible molecular tools. Here, we describe an enhanced Labeling Immune Partnerships by SorTagging Intercellular Contacts (LIPSTIC) platform utilizing the immunologically inert human CD58/CD2 receptor-ligand pair (SrtA-hCD58/G5-hCD2). This system provides a modular alternative to previous LIPSTIC animal models and enables the detection and recovery of T cells following Ag-dependent interactions with distinct APC populations in vitro and in vivo. We applied this approach to interrogate the role of type 3 innate lymphoid cells (ILC3s), an unconventional ROR{gamma}t Ag-presenting population whose role in shaping Ag-specific T cells in vivo remain poorly defined. Modified LIPSTIC successfully recorded cognate interactions between ILC3s and naive CD4 T cells in the spleen and enabled direct comparison with CD4+ T cells activated by conventional dendritic cells (cDCs). Although interactions with ILC3s resulted in lower frequency of in vivo-labeled T cells than cDC2s, CD4+ T cells showed comparable cell division and expression of activation markers irrespectively of an APC type. Together, these findings establish SrtA-hCD58/G5-hCD2 as a versatile LIPSTIC receptor-ligand pair for tracking cell-cell interactions in vivo and provide a robust tool for studying how APC identity influences Ag-specific T-cell responses.

Studying algae requires specialized equipment to characterize culture growth and physiological performance and non-invasive monitoring of these cultures typically relies on optical density (OD) measurements. Currently, OD is often measured using standard spectrometers that suffer from detector saturation at high cell densities, which requires time consuming sample dilutions and lacks compatibility with flat-walled cell culture flasks like Roux bottles. Here we describe a 3D-printable device to measure optically dense algal cultures directly within these cultivation vessels. This instrument combines light attenuation and scattering, resulting in a dual-sensor system that utilizes near-infrared light (950 nm) to precisely quantify biomass without interference from changing photosynthetic pigments. Cultures that reach high cell densities can be assumed to be accurately quantified in situ using the scattered signal, preserving the integrity of the culture and eliminating the need for subsampling. This method is adaptable to the most common geometries of Roux bottles (T-25, T-75, and T-175) and could be used to monitor small-scale batch cultures without contamination risks. A cyanobacteria algal species (Limnospira platensis) and three flask sizes were analysed on light attenuation linearity and scattering detection limits at different cell concentration, demonstrating that the light attenuation module can reliably measure biomass up to an OD of 3.1 (equivalent to >1.5 g/L) without requiring dilution.

Human rhinovirus (HRV), a major cause of upper respiratory infections, has increasingly been associated with lower respiratory tract disease. Although tools to investigate HRV pathogenesis are needed, they remain limited. Reporter viruses expressing fluorescent or luminescent proteins are powerful tools for analyzing viral distribution and replication; however, insertion of a reporter gene often impairs viral replication, and the inserted gene is readily lost during serial passage. To address these problems, we established a versatile and stable strategy for generating reporter HRV and other enteroviruses. The gene encoding mScarlet3, a red fluorescent protein, was inserted between the 5'UTR and VP4 gene, a site previously used in other enteroviruses but not in HRV. Optimizing the 2A cleavage sequence between mScarlet3 and VP4 yielded a reporter HRV with growth kinetics comparable to those of the wild-type virus. To improve the genetic stability of the reporter insert, we evaluated GC-content optimization of the reporter gene and relocation of the cis-acting replication element (CRE). GC-content optimization substantially improved reporter stability in both HRV-B14 and EV-A71, whereas CRE relocation provided additional stabilization in EV-A71 but not in HRV-B14, demonstrating the potential applicability of this strategy to other enteroviruses. This strategy for constructing stable reporter HRV and other enteroviruses will facilitate studies of enterovirus pathogenesis.

Objective: Hyperthyroidism and hypothyroidism related to iodine deficiency are major public health concerns in Afghanistan. This study aimed to assess the frequency of thyroid dysfunction among male patients referred for thyroid testing and its association with age, and to examine monthly trends in thyroid dysfunction at Asia Med Laboratory in Herat, Afghanistan, from July 2021 to January 2022. Methods: A retrospective analysis was conducted on 250 male patients aged 0-69 years. We measured Serum TSH, total T4, and total T3 levels, and thyroid status was classified using age specific reference ranges. In addition, the frequency of thyroid dysfunction was analyzed across age groups with monthly trends of thyroid state. Results: Overall, the euthyroid state consisted of 69.2% of participants, 24.8% with overt hypothyroidism, 3.2% with overt hyperthyroidism, and 2.8% with subclinical hyperthyroidism. Thyroid status differed significantly by age (p = 0.0135), with hypothyroidism increasing in older age groups and reaching its highest proportion among men aged 60-69 years (55.6%). Euthyroidism predominated in patients aged 10-39 years, while hyperthyroidism across age groups remained relatively infrequent. After September 2021, a threefold increase was observed in the total number of male patients referred for thyroid testing. During this period, the proportion of hyperthyroidism increased slightly, whereas hypothyroidism cases declined. Conclusion: In conclusion, hypothyroidism was more frequent with older age. The rise in absolute case numbers after September 2021 likely reflects increased patient referrals, underscoring the need for ongoing monitoring of thyroid function. The study may assist in the early management of thyroid disorders and in reducing their complications.

Gray leaf spot can cause severe turfgrass damage, particularly in intensively managed sports fields, while disease management remains challenging due to the rapid development of symptoms under favorable environmental conditions. Early detection of Pyricularia oryzae could therefore provide valuable information for anticipating disease development and supporting management decisions. This study aimed to develop a digital PCR (dPCR) molecular tool to support the monitoring and management of gray leaf spot disease in stadium turfgrass. The assay showed high sensitivity, consistently detecting P. oryzae when infected tissue represented only 0.05% of a mixture with healthy turfgrass, and enabled pathogen detection before symptom appearance under both controlled and field conditions. Different matrices were also investigated across 15 stadiums to identify the most relevant material for disease monitoring. Roots showed the highest prevalence of P. oryzae detection, including during asymptomatic periods, highlighting their potential as a relevant matrix for pathogen surveillance. In a stadium monitored several months after a gray leaf spot outbreak, P. oryzae DNA was still detected in roots from 27 of the 32 sampled zones despite the absence of visible symptoms. Overall, these results demonstrate the potential of dPCR for the early detection and monitoring of P. oryzae in stadium turfgrass, particularly through root sampling during periods when environmental conditions are not favorable for gray leaf spot development, and a spatio-temporal surveillance at stadium scale.

Broad-range RT-qPCR assays are essential for detecting highly diverse viruses within virus families, but designing suitable primers remains challenging because of extensive sequence diversity and biases in public sequence databases. Here, we present a semi-automated workflow that combines sequence clustering and varVAMP to streamline pan-RT-qPCR primer design. The workflow reduces sequence redundancy, improves the representation of viral diversity, and facilitates the identification of broadly reactive primer candidates. We demonstrate the workflow using the arboviral genera Orthoflavivirus and Alphavirus as proof-of-concept datasets, showing its applicability to large and genetically diverse viral sequence collections. The workflow provides a practical, scalable, and reproducible framework for developing broad-range primer candidates and may support surveillance, diagnostics, and preparedness for emerging or divergent viruses.

Soil nematodes play key roles in regulating organic matter decomposition, nutrient cycling and trophic interactions in the soil ecosystem, and are very useful bioindicators of soil condition and soil health. But to characterise them, we need reliable measurements of nematode biodiversity in soils. DNA-based techniques such as metabarcoding have recently explored their biodiversity, with successful results. However, there are many methodological steps that can modify the outcome considerably and require informed decisions. In this study, we tested the effect on nematode alpha and beta diversity metrics of three methodological steps, namely i) type of extraction of genetic material from the matrix, either from soil directly with different starting amount of material or extracting nematodes first via the Baermann or Oostenbrink methods, ii) the choice of taxonomically-relevant DNA segment for amplification, with a nematode specific primer (NemF/18Sr2b) and a universal eukaryotic primer (3NDf/1132rmod), both within 18S rDNA, and iii) the selection of appropriate reference library for taxonomic assignment (comparing three nematode specialised databases and three broad taxonomic databases). Observed differences in alpha diversity were more pronounced when comparing the nematode extraction vs soil extractions. In particular, direct soil extractions co-extracted many non-nematode taxa, especially if amplified with a universal primer, which can significantly reduce the number of nematode reads obtained and therefore the accuracy of nematode community indexes and other nematode-based metrics. The choice of the reference database strongly affected the taxonomic identification, which calls for extreme caution when making this selection. Notably, specialised databases tended to over assign reads to nematodes that were detected as non-nematodes by broad taxonomic databases. However, beta diversity metrics were consistent across methods, highlighting these metrics as robust against methodological choices. Our study clearly demonstrates that the choices of methods for nematode biodiversity studies using molecular tools can have a big impact on the results, and decisions should be made consciously and carefully.

GeneCapture is developing a novel, point-of-care, rapid AST technique that, when fully automated, can accomplish AST within 2 hours directly from a raw sample, representing a significant advancement in diagnostic capabilities. For this study, a panel of uropathogen isolates was collected from clinical specimens positive for urinary tract infection from various laboratories in the southern US. A selection of these isolates were then spiked into fresh clean-catch urine samples to produce mock samples covering a variety of antibiotic responses. Testing was performed on 20 strains of Escherichia coli, the cause of over 80% of UTIs, and 6 strains each of Klebsiella pneumoniae and Proteus mirabilis, the next most common causes. Each strain was tested against the susceptible, intermediate and resistant (S-I-R) breakpoints of up to 5 appropriate antibiotics including Amoxicillin-Clavulanate, Cefpodoxime, Ciprofloxacin, Nitrofurantoin and Trimethoprim-Sulfamethoxazole. The new method uses a phenotypic assessment of bacterial growth, comparing responses from bacteria treated with antibiotics at S-I-R breakpoint concentrations to untreated controls as the metric for determining susceptibility. Exposure to the drugs for only 90 minutes gave a Categorical Agreement of 98% to gold standard culture results across the resulting 247 bacteria/antibiotic pairs. Testing on separate days of nine of these strains in triplicate gave a reproducibility of 97%. The sequential steps of this novel AST - sample processing, growth, labeling, and counting - can readily be performed on an automated cartridge that is currently under development. The presented work, which took place in a laboratory setting, demonstrates the efficacy of the assay.

Suspension mass cytometry can measure 50 or more markers per cell and is not impacted by cellular autofluorescence, making it an appealing platform for comprehensive phenotyping of single cells in tissue with high innate fluorescence, such as brain. However, this organ is challenging to reduce to a single cell suspension: it is a deeply interconnected tissue rich in extracellular material and contains a wide variety of cell types including neurons, their supporting glia, and hematopoietic cells from peripheral circulation, any or all of which may have competing requirements for survival through the dissociation process. This study compares approaches for producing single-cell preparations from white and gray matter enriched regions of adult mouse brain, testing simple mechanical dissociation, Accutase, and three gentle enzymes plus DNase: papain, collagenase II, and collagenase plus dispase, all with or without Percoll gradient centrifugation to separate intact cells from debris. Live cells were counted by hemocytometer and Trypan blue exclusion to assess yield and compare across enzyme conditions. Mass cytometry was used to assess cellular composition after representative dissociation, debris removal, and viable cryopreservation. Key proteins measured included beta III-tubulin for neurons, glial fibrillary acidic protein (GFAP) for astrocytes, oligodendrocyte transcription factor (OLIG2) for oligodendrocytes, CD31 for endothelial cells, CD24 for ependymal cells, purinergic receptor P2Y12 (P2RY12) for microglia, and CD45 for leukocytes. Mechanical disaggregation paired with enzymatic dissociation by papain, Collagenase/Disapse, or Collagenase II plus DNase produced approximately 400,000 live cells per 100 grams of tissue. Cell yield was not significantly different between conditions and yields were similar between white and grey matter. Addition of Percoll gradient centrifugation substantially reduced the number of viable cells recovered (average decrease of 88%) and resulted in viable cell numbers that were insufficient for robust analysis of less common cell subsets. In addition to neurons, mass cytometry identified six major populations of non-neuronal cells: oligodendrocytes, astrocytes, endothelial cells, ependymal cells, microglia, and leukocytes. The relative abundance of these six populations was generally unchanged by cryopreservation in both grey matter (r = 0.95), and white matter (r = 0.90), though loss of oligodendrocytes did occur in both tissues. Proliferation, as measured by Ki67 positivity, was non-existent; consistent with this, levels of phosphorylated STAT1 (p-STAT1) and p-STAT3 proteins were low in all cell populations and unchanged by cryopreservation. A modest increase in p-S6(S235/236) after cryopreservation was consistently observed only in microglia. Together, these results establish standardized procedures for generating viable single cell suspensions for cytometry that preserve the cellular diversity of the adult murine brain with minimal perturbation of intracellular signaling cascades.

Protein aggregates are a diverse array of intra- and extracellular structures containing misfolded proteins with limited macromolecular movement. Protein aggregation is induced by aging and cellular stress and is a hallmark of neurodegenerative diseases including Huntington's, Alzheimer's, and Parkinson's. Fluorescent tools to visualize global protein aggregation in whole organisms are, therefore, important for screening genes and compounds that may reduce or alter protein aggregation in connection with improved health outcomes. Fluorescent probes that report low fluidity structures in cells, such as molecular rotor dyes, are gaining popularity and overcome certain limitations of targeted markers such as tagged proteins or antibodies. However, permeability has been a key barrier preventing widespread use of such probes in whole organisms, including in C. elegans. To achieve visualization of non-specific protein aggregates in whole C. elegans nematodes without the need for strain engineering, we have developed a simple soaking protocol. Our protocol overcomes the permeability limitations of the commercially available molecular rotor dye, Proteostat, enabling widespread labeling of protein aggregates in live and post-fixed worms. Here, we present our novel approach and demonstrate quantification of protein aggregates in multiple tissues to detect differences in protein aggregation due to both genotype and treatment effects. We also outline anticipated future applications for this protocol, including its adaptation for other dyes, as well as limitations and special considerations for its use.