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Past Issue in 2025
Volume: 15, Issue: 14
Biochemistry
Western Blotting and Immunoprecipitation of Native Human PIEZO1 Channels
PIEZO1 is a mechanically activated ion channel essential for mechanotransduction and downstream signaling in almost all organ systems. Western blotting is commonly used to study the expression, stability, and post-translational modifications of proteins. However, as a large transmembrane protein, PIEZO1 contains extensive hydrophobic regions and undergoes post-translational modifications that increase its propensity for nonspecific protein–protein interactions. As a result, conventional sample preparation methods seem unsuitable for PIEZO1. For example, heating and sonicating transmembrane proteins exposes hydrophobic regions, leading to aggregation, improper detergent interactions, and loss of solubility, ultimately compromising their detection in western blots. To address these challenges, we developed a western blot protocol optimized for human PIEZO1 by preparing lysates consistently at lower temperatures and incorporating strong reducing and alkylation reagents into the western blot lysis buffer to ensure proper protein solubilization and minimal cross-linking. Using the same antibody, we also developed an immunoprecipitation protocol with optimized detergents to maintain the solubilization of native human PIEZO1, enabling the discovery of a new family of auxiliary subunits.
A Fluorescence-Based Flippase Assay to Monitor Lipid Transport by Drs2-Cdc50
Flippases, a functionally distinct group of transmembrane proteins that flip lipids from the extracellular or luminal side to the cytosolic side of biological membranes, are key players in many important physiological processes, such as membrane trafficking and cellular signaling. To study the function of these membrane proteins under chemically defined conditions, reconstituting them into artificial vesicles is a crucial and effective approach. There are various methods for protein reconstitution involving different detergents and detergent removal techniques to integrate membrane proteins into artificial vesicles. In this protocol, we describe the reconstitution of the yeast flippase complex Drs2-Cdc50, which translocates phosphatidylserine across membranes of the trans-Golgi network at the expense of ATP hydrolysis. The flippase complex is incorporated into liposomes using a zwitterionic detergent, followed by detergent removal via dialysis—a gentle and effective strategy that helps preserve protein function. To evaluate the activity of the reconstituted flippase complex, two complementary assays are employed: (1) a fluorescence-based quenching assay to measure lipid transport; and (2) an ATPase assay using an ATP-regenerating system to measure ATP hydrolysis. Together, these methods provide a robust platform for analyzing the functional reconstitution of Drs2-Cdc50 in a defined membrane environment.
ZnCl2 Precipitation-Assisted Sample Preparation for Proteomic Analysis
This manuscript details protocols for the ZnCl2 precipitation-assisted sample preparation (ZASP) for proteomic analysis. By inducing protein precipitation with ZASP precipitation buffer (ZPB, final concentration of ZnCl 2 at 100 mM and 50% methanol), ZASP depletes harsh detergents and impurities, such as sodium dodecyl sulfate (SDS), Triton X-100, and urea, at high concentrations in solution from protein solutions prior to trypsin digestion. It is a practical, robust, and cost-effective approach for proteomic sample preparation. It has been observed that 90.2% of the proteins can be recovered from lysates by incubating with an equal volume of ZPB at room temperature for 10 min. In 1 h of data-dependent acquisition (DDA) analysis on an Exploris 480, 4,037 proteins and 25,626 peptides were quantified from 1 μg of mouse small intestine proteins, reaching a peak of 4,500 proteins and up to 30,000 peptides with 5 μg of input. Additionally, ZASP outperformed other common sample preparation methods such as sodium deoxycholate (SDC)-based in-solution digestion, acetone precipitation, filter-aided sample preparation (FASP), and single-pot, solid-phase-enhanced sample preparation (SP3). It demonstrated superior performance in protein (4,456 proteins) and peptide identification (29,871 peptides), lower missing cleavage rates (16.3%), and high reproducibility (Pearson correlation coefficients of 0.96 between replicates) with similar protein distributions and cellular localization patterns. Significantly, the cost of ZASP per sample with 100 μg of protein as input is lower than 30 RMB, including the expense of trypsin.
Bioinformatics and Computational Biology
Fast TV-PRO-seq: Accelerated and Streamlined Protocol for Timing RNA Polymerase Pausing
Transcriptional pausing dynamically regulates spatiotemporal gene expression during cellular differentiation, development, and environmental adaptation. Precise measurement of pausing duration, a critical parameter in transcriptional control, has been challenging due to limitations in resolution and confounding factors. We introduce Fast TV-PRO-seq, an optimized protocol built on time-variant precision run-on sequencing (TV-PRO-seq), which enables genome-wide, single-base resolution mapping of RNA polymerase II pausing times. Unlike standard PRO-seq, Fast TV-PRO-seq employs sarkosyl-free biotin-NTP run-on with time gradients and integrates on-bead enzymatic reactions to streamline workflows. Key improvements include (1) reducing experimental time from 4 to 2 days, (2) reducing cell input requirements, and (3) improved process efficiency and simplified command-line operations through the use of bash scripts.
Biological Engineering
Protocol for 3D Bioprinting a Co-culture Skin Model Using a Natural Fibrin-Based Bioink as an Infection Model
The skin microbiome, a diverse community of microorganisms, plays a crucial role in maintaining skin health and homeostasis. Traditional studies have relied on two-dimensional (2D) models, which fail to recreate the complex three-dimensional (3D) architecture and cellular interactions of in vivo human skin, and animal models, which have species-specific physiology and accompanying ethical concerns. Consequently, both types of models fall short in accurately replicating skin physiology and understanding its complex microbial interactions. Three-dimensional bioprinting, an advanced tissue engineering technology, addresses these limitations by creating custom-designed tissue scaffolds using biomaterial-based bioinks containing living cells. This approach provides a more physiologically relevant 3D structure and microenvironment, allowing the incorporation of microbial communities to better reflect in vivo conditions. Here, we present a protocol for 3D bioprinting an in vitro skin infection model by co-culturing human keratinocytes and dermal fibroblasts in a high-viscosity, fibrin-based bioink to mimic the dermis and epidermis. The bioprinted skin tissue was co-infected with Staphylococcus aureus and Staphylococcus epidermidis to mimic bacterial skin disease. Bacterial survival was assessed through colony-forming unit enumeration. By incorporating bacteria, this protocol offers the potential to serve as a more representative in vivo 3D bioprinted skin infection model, providing a platform to study host–microbe interactions, immune responses, and the development of antimicrobial therapeutics.
Biophysics
Workflow for Fluorescence-Targeted Lamella Milling From Vitrified Cells With a Coincident Fluorescence, Electron, and Ion Beam Microscope
Cryo-electron tomography (cryo-ET) is the main technique to image the structure of biological macromolecules inside their cellular environment. The samples for cryo-ET must be thinner than 200 nm, which is not compatible with micron-sized cells. A focused ion beam (FIB), in conjunction with a scanning electron microscope (SEM) to navigate the sample, can be used to ablate material from vitrified cells such that a thin lamella remains. However, the preparation of lamellae with a FIB-SEM is blind to the location of specific cellular structures and biomolecules. Furthermore, the thickness and uniformity of lamella, while crucial for high-quality tomograms, cannot be established accurately with the FIB-SEM. These limitations strongly affect the success rate for cryo-ET on FIB-milled lamellae and thereby the total throughput of the workflow. To mitigate these problems, a coincident light, electron, and ion beam cryo-microscope was developed by retrofitting a fluorescence microscope, cryogenic microcooler, and piezo stage on a FIB-SEM. The fluorescence of molecules of interest can be monitored in real time while milling to ensure the final lamella contains the structure of interest. In addition, reflected light microscopy can be used for thickness and quality control of the lamella. In this protocol, we will describe how the coincident microscope can be used to prepare lamellae from vitrified cells.
Cell Biology
Quantifying Intracellular Distributions of HaloTag-Labeled Proteins With SDS-PAGE and Epifluorescence Microscopy
Counting protein molecules helps reveal the organization of components within cellular structures and the stoichiometries of protein complexes. Existing protein and peptide quantitation methods vary in their complexity. Here, we report a straightforward workflow to measure the absolute number of HaloTag-labeled myosin 10 (Myo10) molecules in U2OS cells. Myo10 is a motor protein that plays a prominent role in cellular protrusion formation. Various biochemical and biological properties of Myo10 are established, but it is not well-defined how many molecules of Myo10 pack into narrow cellular structures called filopodia. We present a workflow for using SDS-PAGE to calibrate Myo10 signal with a reference protein, segmenting epifluorescence microscopy images to map Myo10 intracellular distribution, and interpreting the results to derive biological and functional insights. Our protocol is simple to employ and not only applicable for Myo10 research but also easily adaptable for other biological systems that use HaloTag.
Fixation and Expansion Microscopy of Xenopus Egg Extract Spindles
In vitro systems based on Xenopus egg extracts have elucidated many aspects of spindle assembly. Still, numerous unknowns remain, particularly concerning the variation in spindle morphologies. The X. laevis and X. tropicalis egg extract systems, which recapitulate diverse spindle sizes and architectures, serve as ideal tools to investigate the regulation of spindle morphometrics. However, fully understanding spindle architectural differences is hindered by the spindle's size and high microtubule density. Indeed, classical fluorescence microscopy lacks the resolution to detail the organization of spindle microtubules, and although electron tomography can distinguish individual microtubules, segmenting thousands of microtubules and tracking them across dozens of sections remains an unachieved challenge. Therefore, we set out to apply expansion microscopy to the study of Xenopus egg extract spindles. During this process, we realized that optimizing spindle fixation as well was crucial to preserve microtubule integrity. Here, we present an optimized fixation and expansion microscopy protocol that enables the study of spindle architecture in egg extracts of both X. laevis and X. tropicalis. Our method retains the fluorescence of rhodamine tubulins added to the extracts and allows for both pre- and post-expansion immunofluorescence analysis.
Using Beads as a Focus Fiduciary to Aid Software-Based Autofocus Accuracy in Microscopy
Brightfield microscopy is an ideal application for studying live cell systems in a minimally invasive manner. This is advantageous in long-term experiments to study dynamic cellular processes such as stress response. Depending on the sample type and preparation, the inherent qualities of brightfield microscopy, being very low contrast, can contribute to technical issues such as focal drift, sequencing lags, and complete failure of software autofocus systems. Here, we describe the use of microbeads as a focus aid for long-term live cell imaging to address these autofocus issues. This protocol is inexpensive to implement, without extensive additional sample preparation, and can be used to capture focused images of transparent cells in a label-free manner. To validate this protocol, a widefield inverted microscope was used with software-based autofocus to image overnight in time-lapse format, demonstrating the use of the beads to prevent focal drift in long-term experiments. This improves autofocus accuracy on relatively inexpensive microscopes without using hardware-based focus aids. To validate this protocol, the KNIME logistics software was used to train a random forest model to perform binary image classification.
Microbiology
Inducible HIV-1 Reservoir Reduction Assay (HIVRRA), a Fast and Sensitive Assay to Test Cytotoxicity and Potency of Cure Strategies to Reduce the Replication-Competent HIV-1 Reservoir in Ex Vivo PBMCs
The HIV-1 reservoir, consisting of transcriptionally silent integrated HIV-1 proviruses, is a major barrier to a cure, as it persists during effective antiretroviral therapy (ART) and is the source of viral rebound upon treatment interruption. Some of the strategies explored for HIV cure focus on the identification of compounds to either reactivate and eliminate the HIV reservoir (“shock and kill”) or to prevent HIV reservoir reactivation and induce deep proviral latency (“block and lock”). Paramount in developing these HIV-1 cure strategies is determining the effect of the compounds on the size of the inducible HIV-1 reservoir in blood from people living with HIV-1 (PWH). Traditionally, viral outgrowth assays have been the primary method to determine the inducible HIV-1 reservoir in CD4+ T cells from PWH. However, these assays are labor-intensive, time-consuming, and often have low sensitivity. We have recently developed the inducible HIV-1 reservoir reduction assay (HIVRRA), a rapid, cost-effective, and sensitive method to measure the impact of compounds on the inducible replication-competent HIV-1 reservoir in total peripheral blood mononuclear cells (PBMCs) from PWH ex vivo. The HIVRRA simultaneously evaluates the effect of test conditions on the size of the inducible replication-competent HIV-1 reservoir as well as the specificity and toxicity of the test strategy. Using total PBMCs instead of purified CD4+ T cells reduces processing time and resource requirements. This makes the HIVRRA a more practical, scalable tool for evaluating potential HIV-1 cure strategies.
Metabolite Production and Extraction of Indole Compound From the Tomato Endophyte Streptomyces sp. VITGV100
Endophytic actinomycetes, particularly Streptomyces species, have gained significant attention due to their potential to produce novel bioactive compounds. In this study, we isolated and characterized an endophytic Streptomyces sp. VITGV100 from the tomato plant (Lycopersicon esculentum), employing the direct streak method and whole-genome sequencing. A genome analysis was done to uncover its biosynthetic potential and identify indole-type compounds. The strain's secondary metabolite production was evaluated through GC–MS analysis, and its antimicrobial activity was tested against selected human pathogenic bacteria. Our protocol outlines a comprehensive approach, describing the isolation and extraction of metabolites and genome mining for indole-type compounds. This isolate has potential pharmaceutical applications, accelerating the discovery of novel indole-type bioactive compounds.
Flow Cytometric Quantification of HIV-1-Infected Cells Expressing Either Abortive or Elongated HIV-1 Transcripts Using Flow-FISH
The persistence of the HIV-1 reservoir remains the ultimate obstacle in achieving a cure. Cure strategies targeting the HIV-1 reservoir are under development, and therefore, finding ways to improve the detection of the reservoir is crucial. Several reservoir detection techniques exist to assess different markers of the HIV-1 reservoir, such as PCR-based assays and protein-based flow cytometric methods. We developed a flow cytometry-fluorescent in situ hybridization (flow-FISH) approach that assesses HIV-1 at the transcriptional level. Using a combination of probes that target either the HIV-1 trans-activation response (TAR) region and 5′ long terminal repeat (LTR) or the Gag sequence, our assay distinguishes between infected cells expressing abortive or elongated HIV-1 RNAs. This assay utilizes the branched-DNA method to amplify the fluorescent signal of the hybridized RNA probes and can be used directly for thawed or cultured cells, with the option to include surface antibody staining. Cellular expression of abortive and/or Gag HIV-1 RNAs is measured by flow cytometry. Our flow-FISH approach gives insight into the transcriptional dynamics of the HIV-1 reservoir and allows for the characterization of latently infected cells.
Thermus thermophilus CRISPR Cas6 Heterologous Expression and Purification
The CRISPR-Cas system of Thermus thermophilus has emerged as a potent biotechnological tool, particularly its Cas6 endonuclease, which plays a crucial role in CRISPR RNA (crRNA) maturation. This protocol details a robust and reproducible method for the high-level expression and purification of recombinant T. thermophilus Cas6 proteins (Cas6-1 and Cas6-2) in E. coli. We describe a streamlined approach encompassing plasmid construction using seamless assembly, optimized bacterial heterologous expression, and multi-step purification leveraging affinity and size-exclusion chromatography. The protocol outlines the generation of both His-tagged and GST-tagged Cas6 variants, enabling flexibility in downstream applications. Key steps, including primer design, PCR optimization, competent cell transformation, and chromatography strategies, are meticulously detailed with critical parameters and troubleshooting guidance to ensure experimental success and high yields of highly pure and active T. thermophilus Cas6 proteins. This protocol is useful for researchers requiring purified T. thermophilus Cas6 for structural studies, biochemical characterization, and the development of CRISPR-based biotechnological tools.
Comprehensive Mapping of EZ-Tn5 Transposon Insertion Sites in Pseudomonas argentinensis SA190 Using RATE-PCR
Transposon mutagenesis is a powerful tool for investigating gene function in bacteria, particularly in newly discovered species. In this study, we applied the hyperactive EZ-Tn5 transposase system to Pseudomonas argentinensis SA190, an endophytic bacterium known for enhancing plant resilience under drought stress. By leveraging the random amplification of transposon ends (RATE)-PCR method, we successfully mapped the insertion sites of the transposon within the SA190 genome. This approach enabled the precise identification of disrupted genes, offering insights into their roles in bacterial function and interaction with host plants. Our comprehensive protocol, including competent cell preparation, transformation, and insertion site mapping, provides a reliable framework for future studies aiming to explore gene function through mutagenesis.
Molecular Biology
Evaluation of Translation Rate Through L-azidohomoalanine (AHA) Incorporation and Subsequent Alkyne Fluorophore–Mediated Click Chemistry in Yeast
Accurate measurement of protein translation rates is crucial for understanding cellular processes and disease mechanisms. However, existing methods for quantifying translation rates in yeast cells are limited. Here, we present a streamlined protocol for measuring protein translation rates in Saccharomyces cerevisiae using the methionine analog L-azidohomoalanine (AHA), which is the L isoform of this synthetic amino acid, and fluorophore-labeled alkyne dye-based Click chemistry. Our method involves incorporating AHA into newly synthesized proteins, followed by detection using confocal microscopy, flow cytometry, and SDS-PAGE. We validated our protocol by measuring translation rates under various stress conditions, including heat stress, endoplasmic reticulum (ER) stress induced by tunicamycin, and translation inhibition by cycloheximide. Confocal microscopy revealed differential AHA incorporation and fluorescence intensity across conditions. Flow cytometry quantitatively confirmed significant increases in translation rates under heat stress and decreases under ER stress compared to unstressed conditions at 6 and 24 h post-treatment. Imaging of gels under fluorescence detectors following SDS-PAGE further visualized newly synthesized proteins, with no detectable translation after cycloheximide treatment. Our protocol offers enhanced precision and selectivity compared to existing methods for mammalian cells and represents the first standardized approach for measuring translation rates in yeast. Despite limitations in required specialized equipment and expertise, this method holds promise for diverse applications in biotechnology and biomedical research, enabling investigations into protein synthesis regulation in yeast systems.
Multi-phase Training in Virus-Like Particle Synthesis to Foster Science Self-efficacy in Students With Minimal Laboratory Experience
Science self-efficacy describes the confidence individuals have in their ability to accomplish specific scientific practices. Self-efficacy is one factor linked to success and persistence within STEM fields. The purpose of this protocol is to provide research laboratories with effective methods for teaching and mentoring new students in molecular biology, specifically in the synthesis of virus-like particles (VLPs) derived from bacteriophages. VLPs are multivalent nanoparticle structures that can be utilized in multiple biomedical applications, including platforms for vaccine and drug delivery. Production of bacteriophage VLPs using bacterial expression systems is feasible in most laboratory settings. However, synthesizing and characterizing VLPs can be challenging for new researchers, especially those with minimal laboratory experience or a lack of foundational knowledge in molecular biology. To address this, a multi-phase training protocol was implemented to train new students in VLP synthesis, purification, and characterization. This model was optimized for training numerous high school and undergraduate students. By implementing this multi-phase methodology, the students’ confidence in their abilities to perform specific tasks increased and likely enhanced their persistence in STEM.
Fluorescence Polarization-Based High-Throughput Screening Assay for Inhibitors Targeting Cathepsin L
Cathepsin L (CTSL), a lysosomal cysteine protease belonging to the papain-like protease family, is primarily involved in intracellular protein degradation, antigen processing, and extracellular matrix remodeling. It plays critical roles in pathological conditions, including cancer metastasis, neurodegenerative disorders, and viral infection, due to dysregulated activity or overexpression. Thus, inhibitors targeting CTSL are under investigation for therapeutic applications. Current approaches for identifying CTSL inhibitors predominantly rely on fluorescence-labeled substrates, fluorescence resonance energy transfer (FRET), and cell-based screening assays. Here, we applied the principle of fluorescence polarization (FP) to the detection of substrate cleavage activity by CTSL through changes in millipolarization unit (mp) values and established a cost-effective, quantitative, reagent- and time-saving inhibitor high-throughput screening (HTS) assay. We also provide detailed steps for the expression and purification of highly active CTSL from eukaryotic cells, which lays a solid foundation for the FP-based assay. A key advantage of this assay lies in its reduced susceptibility to fluorescence interference, as the fluorescein isothiocyanate (FITC) fluorophore exhibits high quantum efficiency with an emission peak at 535 nm—a wavelength range distinct from most naturally occurring fluorescent molecules. The assay’s adaptability to reaction time, temperature, and dimethyl sulfoxide (DMSO) concentration minimizes false-positive or false-negative results caused by minor experimental inconsistencies, streamlining the screening process. Furthermore, the protocol requires fewer operational steps, reduced incubation time, and lower quantities of CTSL and substrates compared to conventional methods. This rapid, cost-effective, and scalable approach aligns well with the demands of HTS platforms.
Neuroscience
A Novel Composite Method of Post-stroke Epilepsy Induction
The global burden of stroke has increased in the past several decades, and post-stroke epilepsy (PSE) is a common complication. Contrasted with the advancement in knowledge of stroke pathophysiology, the exact pathogenesis of PSE is unclear. Various animal stroke models have been utilized to investigate the underlying mechanisms of PSE, but the success rate of PSE induction is low. To address this limitation, a novel PSE model was established in the rat by inducing status epilepticus using lithium-pilocarpine one week after photothrombotic stroke. Successful indication of status epilepticus and mortality rate at three days after status epilepticus were the main measurements. Potential usefulness of this model was also illustrated by preliminary results on locomotor activity, exploratory behavior, and anxiety level evaluated using the open-field test, as well as mossy fiber sprouting (MFS) in the hippocampal dentate granule cells using Zinc transporter 3 immunofluorescence staining at 8 weeks after PSE induction. This novel composite method of PSE induction may facilitate future studies on the pathogenesis and treatment of PSE.
Plant Science
Virus Isolation and Rice Protoplast Infection
Rice (Oryza sativa), a staple crop sustaining half of humanity’s caloric intake, is threatened by numerous insect-vector-transmitted diseases, such as rice stripe disease, caused by the rice stripe virus (RSV). Most genetic studies on plant antiviral defense mechanisms rely on natural or artificial infection and transgenic approaches, which require months of plant transformation. Here, we present a streamlined protocol that enables rapid analysis of RSV–host interactions within three days. The method encompasses three key phases: (1) polyethylene glycol (PEG)-based precipitation of RSV virions from infected plant tissues, (2) sequential purification through differential ultracentrifugation with glycerol cushion optimization, and (3) high-efficiency transfection of purified virions into rice protoplasts via PEG-mediated delivery. Viral replication is quantitatively assessed using RT-qPCR targeting viral RNA and immunoblotting with RSV nucleocapsid protein-specific monoclonal antibodies. This approach eliminates dependency on stable transgenic lines, allowing the simultaneous introduction of exogenous plasmids for functional studies. Compared with conventional methods requiring several months for transgenic plant generation, our protocol delivers analyzable results within three days, significantly accelerating the exploration of antiviral mechanisms and resistance gene screening.
Evaluating Arabidopsis Primary Root Growth in Response to Osmotic Stress Using an In Vitro Osmotic Gradient Experimental System
The root meristem navigates the highly variable soil environment where water availability limits water absorption, slowing or halting growth. Traditional studies use uniform high osmotic potentials, poorly representing natural conditions where roots gradually encounter increasing osmotic potentials. Uniform high osmotic potentials reduce root growth by inhibiting cell division and shortening mature cell length. This protocol describes a simple and effective in vitro system using a gradient mixer that generates a vertical gradient in an agar gel based on the principle of communicating vessels, exploiting gravity to generate a continuous mannitol concentration gradient (from 0 to 400 mM mannitol) reaching osmotic potentials of -1,2 MPa. It enables long-term Arabidopsis root growth analysis under progressive water deficit, improving phenotyping and molecular studies in soil-like conditions.
Stem Cell
Isolation and Culture of Ferret Airway Stem Cells
Well-differentiated airway epithelial cultures are commonly used to study airway stem cell lineages, ion and fluid transport, respiratory virus infection and replication, and disease mechanisms in vitro. This culture model involves the isolation and expansion of airway stem cells followed by their differentiation at an air–liquid interface (ALI), a process that has been previously documented in humans and mice. Domestic ferrets (Mustela putorius furo) have gained considerable importance in respiratory disease research due to their notable susceptibility to these conditions and their anatomical similarities to humans. Here, we present a comprehensive description of the isolation and culture of stem/progenitor cells from the ferret airway, along with a protocol for their differentiation at the ALI. Our findings have demonstrated that this ferret culture system not only supports the differentiation of the predominant airway epithelial cell types but also facilitates the generation of rare airway epithelial subpopulations, including pulmonary ionocytes, tuft cells, and pulmonary neuroendocrine cells. Additionally, we provide a detailed procedure for measuring transepithelial ion transport relevant to airway diseases, particularly cystic fibrosis. The ability to isolate and culture ferret airway stem cells, combined with ALI differentiation and functional assessment of transepithelial ion transport, offers a powerful platform for evaluating genetic and pharmacologic interventions related to cystic fibrosis.