STAR Protocols

Efficient protein synthesis in eukaryotic cells typically requires a 5' cap structure on messenger RNAs (mRNAs). However, under stress conditions or in viral infection, translation can also occur independently of the cap via internal ribosomal entry sites (IRES). IRES elements are therefore key regulators of protein expression in both viral and cellular contexts. Here we describe a cell-free protocol to quantitatively assess IRES-mediated translation using wheat germ extract (WGE) and a firefly luciferase (FLuc) reporter. The protocol includes template preparation, RNA synthesis and luminescence measurement following in vitro translation in WGE. This method enables rapid and robust comparison of IRES activity under controlled conditions and can additionally be applied to evaluate mRNA modifications designed to enhance translation efficiency. Key featuresO_LIStringent in vitro workflow from DNA template preparation through RNA synthesis and protein synthesis to reporter readout, including quality controls. C_LIO_LIEvaluation of IRES-driven translation suitable for testing combinations of IRES and CDS. C_LIO_LItranslation analysis without radioactive labeling. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/716985v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@417649org.highwire.dtl.DTLVardef@1bcd186org.highwire.dtl.DTLVardef@15fecb3org.highwire.dtl.DTLVardef@acdf8d_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical AbstractPipeline for the production and evaluation of IRES-firefly luciferase constructs using wheat germ extract. (1-4) Preparation: IRES-firefly luciferase constructs are amplified in E. coli and isolated from bacterial cells. Plasmids are linearized to prepare for in vitro transcription. (5-6) Transcript synthesis and verification: In vitro transcription is followed by electrophoretic validation to confirm integrity and correct molecular weight. (7-8) Translation and detection: Translation is executed in wheat germ extract and quantified by measuring reporter activity in a luminometer.

This protocol enables rapid CRISPR-Cas9 genome editing in Saccharomyces cerevisiae by replacing restriction/ligation guide cloning with PCR-based protospacer installation and seamless plasmid recircularization. It describes in silico HDR donor and SgRNA design, install guide sequences into cas9 plasmid by PCR and seamless assembly, plasmid cloning and sequence verification in E. coli, and LiAc/PEG co-transformation of yeast with Cas9-sgRNA plasmid plus HDR donor. The workflow selects yeast colonies on G418 and confirms edits by PCR and sequencing.

Extrachromosomal arrays are unique chromosome-like structures created from DNA injected into the C. elegans germline. Arrays are easy to create and allow for high expression of multiple transgenes. They are, however, unstable unless integrated into a chromosome. Current methods for integration, such as X-rays and CRISPR, damage DNA and are low-efficiency. Here, we demonstrate that the viral integrase PhiC31, which mediates a non-mutagenic recombination between short attB and attP sequences, can be used for extremely efficient and targeted integration of arrays. In this method, a transgene, a selectable marker, and attP sites are injected into the gonad of a strain that (1) has an attB site in its genome, and (2) expresses PhiC31 in its germline. F1 extrachromosomal arrays are cloned, grown for multiple generations with selection, and then screened for homozygous array integrations. The procedure is simple, requires less time than screening for extrachromosomal arrays, and arrays can be screened for transgene function after stable integration. Arrays that transmit are integrated by PhiC31 with 50-95% efficiency, allowing for the isolation of many unique integrants from a single injection. Arrays can also be integrated at fluorescent landing pads and arbitrary sites in the genome. Using nanopore sequencing, we show that three new integrated arrays are between 1.6 and 18 megabases in length, assemble with large repeats, and can contain hundreds of copies of injected transgenes. We have built a collection of strains and plasmids to enable array integration at multiple sites in the genome using various selections. PhiC1-mediated Integration of Arrays of Transgenes (PhiAT) will allow C. elegans researchers to shift from using unstable extrachromosomal arrays to directly integrating arrays.

Nuclei isolation from myelin-rich adult mouse brain regions remains challenging for single-nucleus RNA sequencing because myelin and debris can reduce nuclei quality. We describe an optimized protocol for mouse hippocampi and cerebella using tube-and-pestle homogenization and low-volume sucrose-gradient pelleting with a standard benchtop centrifuge, with optional magnetic enrichment of nuclei to reduce debris/non-nuclear carryover. Under the tested conditions, the workflow produces intact, debris-reduced nuclei and supports downstream 10x Genomics Flex and PARSE WT library preparation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/716374v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@ccbd87org.highwire.dtl.DTLVardef@1aef4bcorg.highwire.dtl.DTLVardef@14569a8org.highwire.dtl.DTLVardef@1bc261_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBenchtop sucrose-gradient pelleting enables rapid nuclei purification from myelin-rich adult mouse brain C_LIO_LIScales across tissue inputs (e.g., hippocampus [~]15-20 mg; cerebellum [~]50-70 mg) without ultracentrifugation or 15 mL gradients C_LIO_LIMagnetic enrichment as the recommended final cleanup step further reduces myelin/debris carryover and is compatible with 10x Flex and PARSE WT workflows. C_LI

Winnie mice are a widely used in vivo model of inflammatory bowel disease carrying a missense mutation in the Muc2 gene. Here, we present a protocol for genotyping Winnie mice using TaqMan allelic discrimination quantitative PCR. We describe tissue collection, rapid crude DNA extraction, probe-based amplification with dual-labeled fluorophores, and fluorescence-based genotype calling in a single reaction. This protocol enables qualitative SNP genotyping without post-amplification processing and can be readily adapted to other defined point mutations. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/704640v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1f5d985org.highwire.dtl.DTLVardef@19bbd34org.highwire.dtl.DTLVardef@1a2d2fcorg.highwire.dtl.DTLVardef@c9baed_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAllelic discrimination qPCR protocol for genotyping the Muc2 p.Cys52Tyr mutation using dual-labeled hydrolysis probes C_LIO_LIEnables rapid discrimination of wild-type, heterozygous, and mutant alleles in a single reaction C_LIO_LICompatible with standard real-time PCR instruments and requires no post-PCR processing C_LIO_LISupports high-throughput genotyping from crude DNA with minimal hands-on time C_LI

SummaryTimeTraits provides a series of functions to enable the extraction of parameters from time series data using Functional Data Analysis methods. We demonstrate its utility in dissecting the changes in curve shape of a circadian clock bioluminescence marker, following a photoperiod shift in wildtype and phyB Arabidopsis. Availability and ImplementationTimeTraits is available in CRAN: https://cran.r-project.org/web/packages/TimeTraits/readme/README.html

Cardiac arrhythmias arise from excitable-media dynamics that are often challenging to teach and visualize in real time. To address this, we developed iMyocyte, an interactive web-based platform for real-time demonstration of excitation wave propagation, conduction block, and reentry dynamics using a simplified cellular automaton model (i.e., a network of discrete cells that change state based on neighbouring cells). In iMyocyte, a network is built from student devices, each representing a model cell coupled to its neighbours to propagate excitation. Unlike traditional teaching materials, iMyocyte lets students manipulate key model parameters and immediately observe how these shape wave dynamics and reentry conditions. Pilot classroom deployments with undergraduate life science students at McGill University indicate high engagement and suggest improved conceptual clarity, motivating future work to improve the platforms usability, robustness, and visualization features, and to conduct formal learning assessment.

IntroductionSuccessful reinnervation following peripheral nerve injury is highly variable, and the molecular programs underlying human muscle degeneration and recovery remain poorly defined. There is a critical need for high-resolution, spatially resolved gene expression data from human skeletal muscle obtained in clinically relevant settings. This study aimed to establish the feasibility of applying spatial transcriptomics to intra-operatively human muscle biopsies and to generate a framework for identifying gene expression signatures associated with reinnervation outcomes. MethodsTo validate the workflow, we collected biopsies intraoperatively from upper-extremity muscles during standard-of-care orthopaedic surgical procedures 5 months after traumatic brachial plexus injury. The flash-frozen biopsy was processed using the 10x Genomics Visium HD high-resolution platform. Quality metrics confirmed high RNA integrity and robust transcript detection at 8 {micro}m resolution. ResultsGenes involved in neuromuscular junction formation, degeneration, and regeneration were identified at subcellular resolution and showed fiber-type-specific expression patterns. Analyses were performed using complementary approaches in Seurat and Loupe Browser. ConclusionsTogether, these findings demonstrate the feasibility of spatial transcriptomics in human muscle, establish baseline gene-expression signatures, and provide a foundation for future studies aimed at identifying biomarkers associated with successful reinnervation and improved nerve-repair strategies.

Traditional nucleic acid extraction methods are costly, lengthy, and highly variable depending on the complexity of the sample matrix or the organism of interest. Workflows may exceed twenty steps, require separate kits for RNA and DNA, and demand expensive instrumentation, creating barriers to both speed and scalability. The AutolabTM HBH system addresses these limitations by using hyperbaric heating (HBH) to achieve temperatures above 100 {degrees}C in a sealed, pressurized environment through induction heating, enabling rapid lysis of diverse organisms and neutralization of macromolecular PCR inhibitors within minutes. The combination of extreme heat and HBH-optimized lyophilized reagents rapidly inactivates nucleases while preserving free nucleic acids. The workflow is streamlined to two steps: heating up to 1 mL of sample in the proprietary HBH bullet, followed by a brief centrifugation to pellet additives. The resulting supernatant is immediately compatible with real-time reverse transcription polymerase chain reaction (RT-PCR) and other downstream molecular assays. Here, we evaluate the systems broad compatibility with diverse sample buffers, matrices, and organisms. Comparative testing was conducted alongside Qiagen extraction methods to benchmark performance.

MOTIVATIONLongitudinal molecular studies of the mouse brain are limited by the need for terminal tissue collection. This prevents analysis of preexisting molecular states and their evolution within the same individual. We developed a stereotactic microbiopsy technique that enables minimally invasive sampling of defined brain regions in vivo. The method preserves survival while yielding material suitable for RNA and nuclei isolation. It provides a practical solution for linking baseline molecular states to subsequent behavioural, pharmacological, or disease-related outcomes. SUMMARYThis study presents a stereotactic microbiopsy technique for sampling defined brain regions in living mice, enabling transcriptomic and epigenomic analyses without sacrificing the animal. The method will allow pre-intervention tissue collection, making it possible to separate preexisting molecular differences from experience- or treatment-induced changes. We show that microbiopsies yield sufficient, high-quality RNA and chromatin for sequencing, with minimal tissue damage that largely resolves over time. The procedure uses standard stereotactic equipment and achieves reproducible spatial precision when the syringe is stabilised. This approach provides a practical framework for within-subject molecular comparisons, reducing animal use and enabling longitudinal profiling of the living mouse brain. It establishes a foundation for investigating how baseline molecular states influence later physiological or behavioural outcomes.

Accurate spectral unmixing is a critical step for flow cytometry data analysis and requires a single stain control for every fluorescent parameter used in an experiment. Currently, compensation particles are often used for making single stain controls when a target protein is of low abundance or a cell type is of low frequency. However, compensation particles introduce incongruencies in emission spectra compared to cells resulting in spectral unmixing or compensation errors. To enable the use of cells regardless of the abundance of target proteins or immune cell type, we generated a bispecific antibody that links a human anti-CD45 and mouse anti-IgG variable region. We refer to this new bispecific tool as CaptureBody (CB) and highlight the benefits of its final nanobody-based design. We provide all sequences and methods necessary for the in-house expression of a CaptureBody to disseminate their use for spectral flow cytometry experiments.

Cellular organelles are not just static structures; they are highly dynamic and directly linked to cellular functions. Changes in their morphology can be early indicators of diseases. Recent advancements in light microscopy techniques have transformed organelle research from qualitative descriptions to precise, quantitative measurements, enabling nanoscale resolution, high-throughput image analysis, and live-cell compatibility. This enables accurate measurement of organelle morphology, dynamics, and spatial organization using modern imaging and analysis techniques. By quantifying organelles, we go beyond simply visualizing to measuring and statistically comparing cellular features across different samples. This protocol addresses a wide range of cellular organelles across all major experimental systems, specifically mentioning mitochondria, myofibers, actin filaments, endoplasmic reticulum, and Golgi apparatus, by integrating experimental design, optimized sample preparation, high-resolution imaging, and validated Fiji/ImageJ-based analysis workflows. For each organelle, step-by-step methods specify reagents, equipment, acquisition parameters, and expected results. While recent advances, such as expansion microscopy, correlative light-electron microscopy, and AI-powered segmentation, offer gains in throughput and resolution, this workflow demonstrates that Fiji-based analysis remains fully capable of delivering high-precision organelle quantification. The entire workflow can be completed within 2-4 weeks, from initial design through validation and the production of measurements suitable for cross-study comparisons. Overall, this protocol establishes a flexible approach to standardize organelle quantification to understand multiple organelles simultaneously in their cellular contexts. Basic Protocol 1: Mitochondrial Quantification Basic Protocol 2: Myofibril Quantification Basic Protocol 3: Golgi Apparatus Morphometry Basic Protocol 4: Endoplasmic Reticulum Network Analysis Alternate Protocol 1: Super-Resolution Imaging Protocol

APOBEC3A catalyzes cytosine-to-uracil deamination in single-stranded DNA and RNA. Physiologically, APOBEC3A functions in innate immunity and aberrant deamination is associated with cytosine mutations in enzymatically preferred YTCW substrate motifs in multiple cancers. Much less is known about the potential contribution of APOBEC3A-catalyzed RNA editing to virus and cancer evolution. Here, we present HAMMER (hairpin-based APOBEC3A-mediated mRNA editing reporter), a rapid luminescence-based cellular assay for measuring RNA editing by APOBEC3A. HAMMER reports APOBEC3A activity as a reduction in the ratio of firefly to renilla luciferase activity. Briefly, tandem renilla and firefly luciferase open reading frames are separated by an optimal APOBEC3A hairpin substrate, in which C-to-U editing of a CGA motif yields a UGA stop codon thus preventing translation of the downstream firefly luciferase reporter, without impacting the upstream renilla reporter. HAMMER activation is dose-responsive, catalytic activity-dependent, and specific to human APOBEC3A. A panel of herpesviral ribonucleotide reductase constructs was used to show that direct inhibition of APOBEC3A results in a dose-responsive recovery of firefly luciferase expression. HAMMER is therefore a scalable and easy-to-use method for quantifying cellular APOBEC3A RNA editing activity and characterizing inhibitors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/695965v2_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@11b1f87org.highwire.dtl.DTLVardef@1b3110dorg.highwire.dtl.DTLVardef@1249c8aorg.highwire.dtl.DTLVardef@a133e4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Functional testing of cytotoxic lymphocytes is essential for research and quality control (QC), but most assays require freshly prepared target cells and extensive handling. A ready-to-thaw, no-wash, flow cytometry-based cytotoxicity assay was developed using pre-labeled K562 targets cryopreserved in STEM-CELLBANKER(R) EX (SCB) as suitably sized aliquots. SCB tolerability was evaluated in K562, NK-92, and primary natural killer (NK) cells; post-cryopreservation label stability of CellTrace Violet (CTV) and carboxyfluorescein succinimidyl ester (CFSE) was assessed; freezing and thawing conditions were optimized; and wash versus no-wash workflows were compared using viability-based and absolute-count readouts, across effector-to-target (E:T) ratios with NK donors and NK-92 cells. Effector viability remained high at SCB concentrations up to 10%, and 5% SCB was selected for assay design. After cryopreservation, CTV labeling remained stable over the tested storage period, whereas CFSE showed substantial signal loss. Warm-medium thawing performed comparably to water-bath thawing, and the consolidated protocol (SCB plus fetal calf serum and thermal buffering) maintained high post-thaw target viability and recovery. In killing assays, lysis increased with increasing E:T ratios; omission of the post-thaw wash had minimal impact, and 5% SCB did not impair cytotoxic function. This ready-to-thaw workflow reduces hands-on time and sample manipulation, while improving standardization for reproducible results and enabling high-throughput functional testing and QC.

DNA-RNA hybrids (R-loops) form transiently on the genome and regulate cellular homeostasis. They also influence genome editing outcomes, highlighting their therapeutic potential in vivo. This protocol enables high-resolution mapping of DNA-RNA hybrids directly from frozen mouse tissues. Following tissue homogenisation and lysis, genomic DNA is extracted, digested and DNA-RNA hybrids are isolated using the hybrid-specific S9.6 monoclonal antibody. The purified hybrids are then processed for whole-genome sequencing to generate R-loop profiles. For complete details on the use and execution of this protocol, please refer to Puzzo, Crossley et al1. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/716701v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1c0e72borg.highwire.dtl.DTLVardef@188cd77org.highwire.dtl.DTLVardef@695c1corg.highwire.dtl.DTLVardef@11e6eff_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOgraphical abstractC_FLOATNO C_FIG

Visualizing protein expression dynamics with high temporal resolution is essential for understanding how cells acquire specific fates and functions during development, where key decisions can occur within minutes. Conventional direct fluorescent tagging often fails to capture these rapid changes in protein expression due to the relatively slow fluorophore maturation time. Indirect epitope-based labeling strategies offer a promising alternative, yet only a limited number of these systems have been developed and used in the context of multicellular organisms. Here, we evaluate and combine four epitope-based indirect labeling systems for live-imaging of proteins in C. elegans: the SunTag, Frankenbody, MoonTag and AlfaTag systems. Each system uses a fluorescently labeled high-affinity single-chain antibody or nanobody to recognize short peptide epitopes fused to a protein of interest, enabling immediate visualization of newly synthesized proteins. We demonstrate that all four systems specifically label epitope-tagged endogenous proteins and show no detectable cross-reactivity when used in dual-color combinations, enabling simultaneous visualization of distinct proteins within the same embryo. In addition, we show that the SunTag system offers three major advantages over direct labeling: earlier detection of proteins, enhanced sensitivity through signal amplification (as illustrated by CAM-1) and less impact on the function (as demonstrated for ERM-1). Together, this expanded toolkit of epitope-based labeling systems offers many new opportunities for visualizing rapid protein dynamics and for dissecting how their dynamics drive cell fate decisions during development. SUMMARYThe development of epitope-labeling systems has improved live-imaging quality of proteins. Unfortunately, limited systems exist for multicellular organisms to study protein expression in the context of development. Here, we expand the epitope-labeling toolbox for C. elegans by combining SunTag or Frankenbody with MoonTag or AlfaTag. Our data indicates that these systems simultaneously visualize different endogenous proteins without cross-reactivity. Moreover, the SunTag system shows advantages over direct labeling: earlier detection, enhanced sensitivity through signal amplification and less impact on protein function. This expanded epitope-labeling toolbox in C. elegans provides opportunities for accurate visualization of different proteins that drive cell fate decisions. O_FIG O_LINKSMALLFIG WIDTH=155 HEIGHT=200 SRC="FIGDIR/small/703904v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@449fe0org.highwire.dtl.DTLVardef@15c68cforg.highwire.dtl.DTLVardef@1e51ff8org.highwire.dtl.DTLVardef@196114d_HPS_FORMAT_FIGEXP M_FIG C_FIG

FSFC is an emerging technology that can greatly enhance our understanding of the single-cell proteomic landscape. However, its application to cells derived from solid tissues has been hampered by their complex autofluorescence signatures and lack of optimized tools for non-immune cells. Here, we present a protocol and discuss key controls that minimize the impact of unmixing errors enabling us to resolve multiple EC subpopulations isolated from different tissues in models of chronic tissue injury. Research Topic(s)Vascular biology, cell heterogeneity, full spectrum flow cytometry Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/695385v2_ufig1.gif" ALT="Figure 1000"> View larger version (43K): org.highwire.dtl.DTLVardef@1745181org.highwire.dtl.DTLVardef@1930db9org.highwire.dtl.DTLVardef@16a0b3dorg.highwire.dtl.DTLVardef@107ec29_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsOptimisation of a FSFC panel to enable in-depth phenotyping of tissue- and model-specific endothelial subpopulations from solid tissues. Discussion of appropriate controls to minimize the impact of tissue autofluorescence and enhance the signal-to-noise ratio for cell phenotyping in complex models of inflammation and fibrosis. Trajectory analysis to track cellular plasticity over time. Application of full spectrum cell sorting to isolate rare endothelial subpopulations with complex phenotypes.

Fiber photometry measures neural activity in vivo from genetically encoded indicators. Recordings generate large datasets that require extensive preprocessing and robust statistical methods for meaningful interpretation. However, existing analysis tools demand programming expertise, limiting accessibility. Here we describe FPmotion, a comprehensive, user-friendly software platform for batchwise processing, integration, and statistical analysis of fiber photometry data with or without accompanying behavioral information. FPmotion performs filtering, isosbestic regression, {Delta}F/F computation, detrending, and z-scoring. It also extracts detailed peak properties at whole-file, block-level, and individual-peak resolution. When behavioral data is provided, FPmotion automatically identifies behavioral bouts, computes bout-level statistics, performs peri-event signal extraction, and supports multi-group comparisons through ANOVA- and LMEM-based statistical frameworks. All analyses generate publication-ready figures and structured CSV outputs suitable for downstream workflows. FPmotion also introduces a dedicated alignment module for peri-event signal analysis. This module applies dynamic time warping followed by barycenter averaging to realign peri-event traces while preserving the behavioral time anchor, producing cleaner and more temporally coherent peri-event motifs. We demonstrate FPmotions capabilities using the dlight1.1 sensor to measure dopamine responses from ventral striatum in behaving mice before and after amphetamine treatment. Together, FPmotion offers a fully automated framework for FP data analysis that improves interpretability and accessibility while reducing analysis time substantially.

Investigating single-cell dynamics and morphology in tissues and embryos requires highly accurate quantitative analysis of microscopy images. Despite significant advances in the field of bioimage analysis, even the most sophisticated segmentation and tracking algorithms inevitably produce errors (e.g. : over segmentation, missing objects, miss-connected objects). Although error rate may be small, their propagation throughout a time-lapse sequence has catastrophic effects on the accuracy of tracking and extraction of single cell parameters. Extracting single cell temporal information in the context of tissue/embryo requires thus expert curation to identify and correct segmentation errors. In the movies commonly used in developmental biology and stem cell research, both the number of imaged cells and the duration of recording are large, making this manual correction task extremely time-consuming. This has now become a major bottleneck in the fields of development, stem cell biology and bioimage analysis. We present here EpiCure (Epithelial Curation), a versatile tool designed to streamline and accelerate manual curation of segmentation and tracking in 2D movies of large epithelial tissues. EpiCure uses temporal information and morphometric parameters to automatically identify segmentation and tracking errors and provides user-friendly tools to correct them. It focuses on ergonomics and offers several visualization options to help navigating in movies of tissue covering a large number of cells, speeding up the detection of errors and their curation. EpiCure is highly interoperable and supports input from a wide range of segmentation tools. It also includes multiple export filters, enabling seamless integration with downstream analysis pipelines. In this paper, using movies from several animal models, we highlight the importance of curating cell segmentation and tracking for accurate downstream analysis, and demonstrate how EpiCure helps the curation process for extracting accurate single cell dynamics and cellular events detection, making it faster and amenable on large dataset.

Low take rates and inter-tumor variability in growth rates can limit the effectiveness of mouse xenograft models when comparing between groups. To address this problem we developed a simple method to compare multiple cell types within a single mixed xenograft. Individual cell lines or clones were transduced with a lentiviral vector that includes a unique PCR tag, allowing the use of qPCR to determine the proportion of each tagged cell type within a mixed xenograft tumor. We generated vectors with six distinct PCR tags, and two different selectable markers, and have optimized the approach for determining their relative proportions within a mix. An initial pre-amplification step is used to increase the amount of material for subsequent qPCR reactions. This also removes the bulk of the genomic DNA, increasing the specificity of the qPCR step. Samples are then used for qPCR with specific pairs of primers that distinguish between each of the individual PCR tags, and the relative proportion of each tag is determined relative to that in the starting mix. We have tested this approach for in vitro growth of mixed cell cultures and in an orthotopic cecal xenograft model using a human colon cancer cell line. Since each individual tumor is initiated with a mix of cells, multiple tumors within a single animal can be analyzed separately, and overall tumor size is not important. Similarly, multiple metastatic lesions from the same animal can be analyzed individually. Thus, each tumor provides a direct comparison between individually tagged cell lines or clones. This low throughput "bar-coding" approach is simple and cost effective and has the potential to reduce the number of animals needed for xenograft experiments.