STAR Protocols

CD8 T cells play a central role in immune responses to infection and cancer. However, the diversity of T cell receptor (TCR) specificities makes it challenging to study the mechanisms that regulate T cell activation, differentiation, and effector function. Beyond TCR transgenic mouse models, various complex genome-editing approaches have been employed to overcome this challenge. However, these strategies are often technically demanding, time-intensive, and difficult to adapt. Investigators who are interested in testing de novo TCRs under their chosen experimental conditions would benefit from a standardized and accessible method. Here, we describe a protocol that combines ribonucleoprotein (RNP)-based CRISPR-Cas9 editing with retroviral transduction to enable efficient genetic manipulation of murine CD8 T cells. We show that T cells engineered via this protocol can be generated at sufficient scale for downstream in vitro assays and in vivo adoptive transfer experiments. We expect this method will be useful for investigators who require a standardized and accessible way to study how TCR specificity impacts CD8 T cell responses.

Three-dimensional (3D) spheroid models have become essential in cancer biology, drug screening, and tissue engineering. However, their small size, fragile structure, and tendency to disintegrate during routine histoprocessing present persistent technical challenges. Conventional paraffin embedding often results in tissue fragmentation, loss of spatial orientation, and poor section quality, whereas cryosectioning often compromises cellular morphology. Here, we present a robust, cost-effective protocol for preserving and sectioning fragile 3D spheroids, resulting in high-quality histological sections with intact architecture and excellent cellular detail. The method involves optimized handling and embedding procedures that stabilize spheroids during standard formalin fixation, paraffin infiltration, and microtomy, eliminating mechanical distortion and preserving spherical integrity for consistent sectioning. We demonstrate successful application across different cell line spheroids, with subsequent compatibility with hematoxylin and eosin (H&E) staining protocols. Compared to conventional methods, our approach significantly reduces sample loss, improves inter-section reproducibility, and preserves fine structural features such as necrotic cores, proliferative zones, and extracellular matrix components. This protocol provides a reliable, accessible solution for routine histological analysis of fragile 3D spheroids, facilitating more accurate morphological and molecular assessment in translational research settings. Key featuresO_LIMaintains spheroid integrity: Prevents mechanical distortion, fragmentation, and loss of spatial orientation during processing. C_LIO_LISignificantly reduces sample loss: Decreases failure rate compared to traditional methods, conserving valuable samples. C_LIO_LIBroad spheroid compatibility: Works effectively with primary tumor-derived, stem cell-derived, and co-culture spheroid models. C_LIO_LIEnables high-quality sectioning and staining: Delivers consistent, reproducible sections that are fully compatible with H&E, IHC, and IF. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/743094v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1670c4org.highwire.dtl.DTLVardef@145810aorg.highwire.dtl.DTLVardef@1accb1org.highwire.dtl.DTLVardef@17481c0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Native polyacrylamide gel electrophoresis is the standard first analytical step after detergent solubilization of thylakoid membranes. It separates photosystem supercomplexes by size and shape, but it forces a polydisperse mixture of protein-detergent particles through a gel of limited pore diameter. Interval-zone free-flow electrophoresis (iZE-FFE) performs that first electrophoretic step in free solution, without ampholytes or immobilines, and returns the complexes as a liquid fraction series ordered by net charge. We describe the operational cycle of iZE, show that bromophenol blue reports the charge density of {beta}-dodecylmaltoside micelles, and apply iZE to Arabidopsis thaliana thylakoid extracts from two sequential solubilization campaigns. After a two-step digitonin then digitonin/{beta}-dodecylmaltoside extraction (experiment 6874), SDS-PAGE of the iZE fractions is crowded: high-molecular mass protein-lipid assemblies of photosystem I and photosystem II release the same subunits into many wells. Native-PAGE of the same fractions resolves ATP-synthase from PSI, a free LHCII pool, anodic PSII, and cathodic PSI, including PSI with and without LHCII. A three-step extraction with a single pH working window (experiment 7154) confirms this charge order. Lowering residual detergent in consecutive extracts still produces partly solubilized high-mass assemblies, whereas a final {beta}-dodecylmaltoside step releases the C2S2M2/C2S2M/C2S2 PSII series and a single PSI band. iZE is therefore a charge-first, orthogonal dimension for native thylakoid biochemistry. Native-PAGE, not SDS-PAGE, is the proper second dimension while solubilization remains incomplete.

Following nervous system injury, surviving bystander neurons may rewire and form synapses with new postsynaptic targets to restore circuit function. This protocol quantifies compensatory motor neuron sprouting at the Drosophila larval neuromuscular junction. We describe larval dissection, immunostaining, Airyscan imaging, image masking, muscle-specific region-of-interest selection, and Fiji-based quantification of Dlg-positive area, bouton number, mean Dlg-positive area per bouton, NMJ arbor length, axonal versus NMJ branch points, ectopic muscle acquisition, and sprouting penetrance. For complete details on the use and execution of this protocol, please refer to Olsen et al.1

Laboratory automation is increasingly being adopted in academic research to improve throughput, reproducibility, and integration with computational workflows. A common bottleneck in molecular biology workflows is the isolation of DNA, typically performed with spin-column kits relying on centrifugation or vacuum. Existing automation solutions depend on magnetic separation systems requiring costly additional modules and specialist kits, making them impractical for laboratories that perform large numbers of extractions only occasionally. Here we present an open-source, 3D-printed vacuum manifold compatible with the Opentrons family of liquid handling robots that enables automated spin-column DNA isolation for up to 24 samples per run (96 with four manifolds in series). The manifold can be manufactured for approximately $2 using standard ABS filament and is compatible with widely available spin-columns, requiring no additional specialist reagents. Using this system, hands-on time for 24 plasmid minipreps was reduced by 80% compared to manual centrifugation and vacuum protocols. The automated workflow demonstrated comparable or greater plasmid yields and eliminated failed recoveries. All design files, labware definitions, and Opentrons protocols are released under the CERN Open Hardware Licence or MIT License. This device lowers the cost barrier to automated DNA purification for academic laboratories worldwide.

Click-qPCR is a browser-based application for relative qPCR analysis that requires a tidy-format CSV file containing four columns: sample, group, gene, and Cq. Preparing this input from qPCR instrument output typically requires manual reformatting and calculation of mean Cq values for technical replicates. To simplify this process, we developed Click-Prep (https://kubo-azu.shinyapps.io/Click-Prep/), an interactive web-based application designed specifically to create Click-qPCR input files. Click-Prep imports CSV, TXT, TSV, and XLS/XLSX files and supports skipping of instrument-generated metadata rows, interactive column mapping, and manual assignment of experimental groups. Users can review technical-replicate measurements, exclude selected rows according to predefined quality-control criteria, and calculate mean Cq values for each sample-group-target combination. Missing or nonnumeric Cq values are flagged for review and must be resolved before the mean is calculated. Click-Prep can also combine compatible formatted CSV files, such as datasets obtained from separate qPCR plates. The resulting dataset is exported as a standardized CSV file containing the four fields required by Click-qPCR. By integrating these operations into a guided browser-based workflow, Click-Prep enables users to prepare Click-qPCR input files rapidly and consistently without programming.

We describe here an efficient method for gene editing in mouse T cells, based on well-established, high-efficiency retroviral transduction protocols. Our platform allows analysis of mutant phenotypes in primary murine T cells in vitro and in vivo. This approach uses a single retroviral vector to simultaneously knockout an endogenous gene and ectopically express its mutant version. This knockout/re-expression vector can be used as the only plasmid to transduce Cas9-expressing T cells, or used together with a Cas9 retroviral vector to transduce T cells from any mouse strain. We validated the system for analysis of murine T cells by targeting key molecules in proximal T cell signaling, i.e. CD3{gamma} and Zap70. We obtain high knockout and re-expression efficiencies in both Cas9-expressing and non-Cas9 T cells. Knockout efficiencies can be further improved by gRNA multiplexing. Endogenous proteins compete with their ectopically expressed mutants or tagged versions for cellular location, protein interactions and cellular functions. Here, we quantified the incorporation of CD3{gamma}-GFP into surface T cell receptor (TCR) complexes. Our data shows that the knockout and re-expression platform improves integration of CD3{gamma}-GFP into the TCR. Therefore, eliminating competition between endogenous and ectopic proteins benefits analyses of protein assemblies and signaling pathways in primary T cells. Furthermore, we validated advantages of our system for mutant analysis using wild-type and mutant Zap70s. Zap70 mutants deficient in TCR binding or kinase activity show their phenotypes only in the absence of endogenous protein, further validating our knockout/re-expression approach. Most importantly, this system can be used to generate gene-edited primary T cells for in vivo studies, such as the quantification of anti-tumor responses. Our knockout and re-expression platform provides a useful gene editing tool for primary T cells in fundamental research and immunotherapy development.

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.

Circular single-stranded DNA (cssDNA) is a versatile biomolecule with applications spanning genome editing, DNA nanotechnology, synthetic biology, molecular diagnostics, and aptamer development. Compared with linear single-stranded DNA, cssDNA offers enhanced structural stability, resistance to in-cellulo degradation by exonucleases and enables the generation of long, sequence-defined DNA molecules that are difficult to obtain through conventional chemical synthesis methods. Despite its growing utility, widespread adoption of cssDNA has been limited by the lack of accessible, scalable, and cost-effective production methods, with many existing workflows relying on specialised reagents, extensive optimisation, or commercially synthesised DNA. Here, we present a streamlined, end-to-end protocol for the laboratory-scale production of high-purity cssDNA using an M13 phagemid-based system and standard molecular biology laboratory infrastructure. The workflow encompasses bacterial culture, phage amplification, nuclease treatment, phage precipitation, anion-exchange purification, and quality control, with practical optimisations to improve yield, reproducibility, and scalability. Using this approach, yields range from 120-195 {micro}g of purified cssDNA from 300 mL of culture supernatant. The protocol provides detailed guidance on critical process parameters, troubleshooting, and quality assessment, enabling reliable production of cssDNA suitable for a wide range of downstream molecular biology and genome engineering applications.

labapi is a Python library that enables computational workflows to connect to LabArchives' electronic lab notebook (ELN). Without an Application Programming Interface (API) connection, researchers must manually add workflow outputs through the LabArchives web interface, navigating to the appropriate page and uploading each output so that it appears with the experimental notes that provide context. labapi translates the flat LabArchives API into a Python object model following the existing hierarchy of the web interface, allowing workflows to navigate and modify notebook content through familiar paths. labapi enables researchers to build interconnected workflows that both read in and write data to LabArchives' ELN automatically. Researchers can inspect those outputs in the notebook, and later analysis code can read them back for another stage of analysis.

Regenerating tissues like the axolotl (Ambystoma mexicanum) limb draw from many cell types, but isolating specific cell populations remains a bottleneck. Standard approaches rely on enzymatic dissociation and fluorescence-activated cell sorting (FACS), which are slow, costly, and require specialized instrumentation. We adapted INTACT (Isolation of Nuclei Tagged in specific Cell Types) to the axolotl, generating a transgenic line (INTACT-olotl) expressing Sun1-sfGFP in limb connective tissue, the population that carries limb positional information, dedifferentiates following injury, and forms the majority of the regenerative blastema. Combining an optimized nuclei isolation protocol with anti-GFP nanobody capture enriched labeled nuclei from ~34.6% of input to 80-89% purity. Relative to dissociation and FACS, this recovers roughly 30-fold more target nuclei in less than half the time and without a cytometer. Bead-bound nuclei are directly applied to combinatorial-barcoding snRNA-seq, eliminating fluorescence-activated nuclei sorting (FANS). Profiling INTACT+ connective tissue during TGF{beta} inhibition revealed a loss of extracellular matrix remodeling with upregulated pro-fibrotic and inflammatory gene programs, resembling an unresolved wound environment. These changes reverse and regeneration resumes upon drug washout, indicating that functional TGF{beta} signaling is required for the transition from wound resolution to blastema formation.

Planning husbandry tasks and experiments with Drosophila melanogaster requires converting a target date into development times that depend on the rearing temperature. This calculation needs to be done for each cross, genotype, and temperature, and the risk of error grows quickly. Available laboratory management tools let users register stocks, crosses, and track them, but they do not create schedules based on a clear, adjustable thermal model. To fill that gap, we developed DrosoTracker, a self-contained web application that works offline and predicts Drosophila development with a thermal summation model recalibrated through regression on data from Powsner (1935) (T0 = 11.78 {degrees}C, DD = 116.38 {degrees}C{middle dot}days, R{superscript 2} = 0.997). The model offers an optional two-level calibration driven by user observations. A wild-type strain first adjusts the model to the laboratorys own conditions. Then each genotype is calibrated against that reference using a random-effects shrinkage estimator that accounts for measurement error and between-batch variability. The model creates schedules for husbandry tasks, evaluates adult cohort survival with the Kaplan-Meier estimator and the log-rank test, and calculates sample size for lifespan studies using Schoenfelds formula. The quantitative components were checked against independent references, including Rs survival package and manual calculations. Ongoing work is focused on validating the calibrated model using cohorts specifically bred for this purpose. DrosoTracker runs entirely in the browser, stores data locally, and is available in English and Spanish.

Background. Reference catalogs such as GENCODE capture most stably expressed mammalian genes but may not include condition-restricted or low-abundance transcripts. Reads supporting unannotated intergenic splice junctions are generally absent from annotation-restricted gene-count matrices; transcript assemblers may reconstruct a subset as novel models, but those models are typically handled separately from the annotated gene-count universe. TxNova is a lightweight command-line tool that directly indexes these unannotated splices from existing BAM files - without an external assembler, gffcompare, or workflow manager - yielding candidate leads for bench validation rather than standalone discovery claims. Methods. TxNova retains CIGAR N junctions from STAR/HISAT2 BAM files that recur in >= 2 samples and are absent from a comprehensive annotation, clusters them into residual loci, and counts each locus alongside annotated genes in a unified matrix. Structure gates - canonical splice motif, same-strand distance, coverage valley, bridging-junction absence, minimum length - remove likely artifacts to yield structure-pass models. An optional contrast filter retains loci detected in treatment but nearly silent in control. Results. A residual splice is a recurrent, unannotated CIGAR N junction that does not overlap any annotated gene body; intronic and antisense channels are out of scope. Across four published mouse treatment arms (GSE221720, GSE166522, GSE157460, GSE193335), harvest catalogs yielded 464, 657, 789, and 594 loci. On GSE221720, 45% of loci (209/464) shared an exact intron with another series, versus 0.074% for excluded junctions; a coordinate-placement control yielded 0/5,000 matches for length-matched intergenic decoys. Masked-gene recovery reached 87.6% (176/201) overall and 99.4% (176/177) among genes with a leak junction - sensitivity rather than precision estimates. An optional contrast provides a presence/absence screen on interval TPM. Residual models are partial reconstructions requiring cloning, RACE, or targeted proteomics for confirmation. Conclusions. TxNova produces a reproducible intergenic residual-locus catalog from existing BAM files, with four downloadable mouse injury/infection catalogs. Cross-series recurrence and coordinate-based null controls support reproducibility above simple placement backgrounds but do not by themselves establish biological validity. The optional contrast step offers a practical screen for candidate leads warranting experimental validation.

To explore adapting qPCR systems for end-point nucleic acid quantification using dyes such as SYTO-9, we quantified serial dilutions of DNA and RNA standards in the range of 0.75 - 200 ng/{micro}l on 384-well qPCR devices. SYTO-9 fluorescence was successfully measured using standard SYBR Green settings. Blank-subtracted relative SYTO-9 signal showed a logarithmic dependence on DNA/RNA concentration (R2 > 0.95). Measurements were highly stable with different incubation times, temperatures of up to 95{degrees}C, and photobleaching. The described approach is a valuable QC option for high-throughput DNA/RNA isolations and could be adapted to additional fluorometric assays beyond nucleic acids.

Double-stranded RNA (dsRNA) is a biologically active contaminant of in vitro transcribed (IVT) mRNA that can reduce protein expression, induce cytokines, affect cell viability, and confound experimental results. Here, we show that dsRNA contaminants containing modified nucleotides are difficult to quantify and functionally assess. Common antibody-based dsRNA ELISAs under-detected pseudouridine- and N1-methylpseudouridine-modified dsRNA, while a higher-sensitivity ELISA improved detection but remained chemistry dependent. Defined dsRNA spike-in experiments showed that effects of dsRNA on cell viability, cytokine induction, and protein expression were chemistry dependent and occurred at low burdens of 0.05-0.5 ng dsRNA per ug RNA. Analytical dsRNA depletion across several workflows did not always predict suppression of cytokine induction, supporting the need for a functional cell-based readout. We engineered B2-S, a single-chain spliced variant of the Flock House virus B2 dsRNA-binding protein, to selectively deplete dsRNA from crude IVT reactions while preserving mRNA recovery and integrity. Among tested methods, B2-S was the only non-enzymatic, non-chromatographic workflow that reduced the cytokine marker IP-10/CXCL10 to mock-transfection levels. These findings establish a paired analytical and functional framework for IVT mRNA quality assessment and introduce B2-S as an accessible method for producing mRNA with improved potency and reduced immunostimulatory activity.

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).

Bone marrow chimeras are widely used to study immune development and function. As the field moves from cesium-137 (137Cs)-based irradiators to X-ray irradiators for safety reasons, it is essential to determine if there are differences in immune system reconstitution after irradiating mice with one of these two radiation sources. Here, we performed a comprehensive immunological comparison of mice lethally irradiated with 137Cs or one of two different X-ray platforms and reconstituted with congenic bone marrow. Mice received 12 Gy total body radiation in two 6 Gy sessions followed by intravenous transfer of donor hematopoietic stem cells and were analyzed eight weeks post-transplant. We assessed mouse survival, donor chimerism, immune cell subset distribution, and activation states across multiple organs (bone marrow, spleen, lymph nodes, liver, and lung). All groups exhibited comparable survival and high levels of donor chimerism, with expected organ-specific reconstitution patterns. Immune lineage distributions, CD4/CD8 ratios, and activation states did not differ by irradiation type. Host-derived radioresistant cells were also similar across all irradiation groups and were predominantly composed of T cells skewed toward an activated phenotype. Overall, our data show that X-ray irradiation with proper filters and energy levels (225 KVp and 320 KVp) can yield equivalent immunological outcomes, including immune reconstitution and activation states, as compared to the same radiation dose from 137Cs-based irradiation in bone marrow chimera models. These results support the continued adoption of X-ray irradiation systems in place of 137Cs for generating bone marrow chimeras to be used across a wide range of immunologic studies.

Introduction. Skeletal muscle loss in sarcopenia and neuromuscular disorders remains a major unmet medical need. AAV9-delivered follistatin (FST), a myostatin/activin antagonist, induces muscle hypertrophy; however, fibre growth without adequate vascular adaptation may limit therapeutic efficacy. We evaluated whether co-administration of a VEGF-A165 plasmid enhances the hypertrophic and angiogenic effects of intramuscular AAV-FST gene transfer in C57BL/6 mice. Methods. Thirty-six C57BL/6 mice (18 males, 18 females) were assigned to PBS vehicle (n=10), AAV-FST (1 x 10^11 vg; n=10), VEGF plasmid (100 ug; n=6), or combination treatment (VEGF plus AAV-FST; n=10). The contralateral hindlimb served as an internal control. Endpoints at Day 115 included hindlimb muscle mass ratio (R/L), transgene expression, FST protein levels, muscle fibre morphometry, capillary density, and safety assessments. Results. Combination therapy produced the highest R/L ratio (1.176 +/- 0.091; p=0.004; d=2.04), whereas AAV-FST alone showed a borderline effect (R/L=1.113; p=0.050). Compared with AAV-FST monotherapy, combination treatment increased muscle FST mRNA approximately 2.1-fold, protein levels approximately 2.0-fold, and the muscle-to-liver expression ratio 2.6-fold. It also induced larger muscle fibres and doubled CD31+ vessel counts versus AAV-FST alone, indicating simultaneous hypertrophy and angiogenesis. No adverse haematological, biochemical, or histopathological findings were observed. Discussion. Combined AAV-FST and VEGF therapy enhanced local muscle hypertrophy, increased capillary density, and improved the muscle-to-liver transgene expression profile compared with AAV-FST monotherapy. The regimen was well tolerated and supports further evaluation of angiogenic preconditioning as a strategy to improve muscle-directed gene therapy for muscle-wasting disorders.

Spatial transcriptomic and proteomic technologies provide complementary insights into tissue organisation, cellular phenotype and function, yet integrating these modalities on the same tissue section remains technically challenging. Sequential workflows must preserve RNA integrity, antigenicity and tissue morphology while maintaining accurate spatial registration. At present, publicly available multimodal datasets suitable for computational method development remain limited. Here, we present a workflow for sequential 10x Genomics Xenium spatial transcriptomics, COMET cyclic immunofluorescence, and haematoxylin and eosin (H&E) histological staining on the same formalin-fixed paraffin-embedded tissue section. We demonstrate this approach across multiple biologically distinct human tissues, including tonsil, hepatocellular adenoma, and matched tumour and non-tumour hepatocellular carcinoma, illustrating the widespread applicability of the workflow beyond a single tissue type. Following image registration, Xenium-derived cell segmentations were applied to protein images to generate integrated single-cell transcriptomic and proteomic measurements for downstream analyses. To facilitate community reuse, we publicly release four representative aligned tissue cores together with transcript coordinates, multiplex protein images, H&E images, cell segmentations, and integrated single-cell datasets. We additionally introduce UnumLocalia, an open-source visualisation and data extraction tool that enables interactive exploration of aligned multimodal images, supports user-defined cell segmentation, and allows export of integrated single-cell data for downstream analyses. Together, this technical protocol, workflow, software, and openly available dataset provide a reusable resource for multimodal spatial biology, supporting advances in biological discovery, computational method development, multimodal data integration, and validation of emerging analytical approaches across complementary spatial technologies.

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.