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Past Issue in 2025
Volume: 15, Issue: 13
Biochemistry
Kinase Mobility Shift Assay (KiMSA) for Assessing Protein Kinase A Activity
The cAMP-dependent protein kinase (PKA) is one of the most extensively distributed kinases among intracellular signal cascades, with a pivotal role in the regulation of various processes, including the capacitation of sperm cells. Traditional assessments of PKA activity rely on the utilization of [γ-32P] ATP and the Kemptide peptide as a substrate. This strategy presents several major drawbacks, including high costs and health risks derived from the manipulation of radioactive isotopes. In this work, we introduce an enhanced non-radioactive assay to quantify PKA activity, termed kinase mobility shift assay (KiMSA), based on the use of a fluorescent-labeled Kemptide (Kemptide-FITC). Once the kinase reaction is terminated, the products can be easily resolved through electrophoresis on an agarose gel and quantified by fluorescence densitometry. We show that KiMSA is suitable for isolated PKA as well as for the enzyme in cell extracts. In addition, it enables quantification of PKA activity during the progression of mouse sperm capacitation. Furthermore, the assay enables monitoring the inhibition of PKA with pharmacological inhibitors in live cells. Therefore, the experimental and optimal assay conditions are set so that KiMSA can be used to assess in vitro as well as in vivo PKA activity in sperm cells. Finally, this method allows for measurement of cAMP concentrations, rendering a versatile technique for the study of cAMP/PKA pathways.
Bioinformatics and Computational Biology
RACE-Nano-Seq: Profiling Transcriptome Diversity of a Genomic Locus
The complexity of the human transcriptome poses significant challenges for complete annotation. Traditional RNA-seq, often limited by sensitivity and short read lengths, is frequently inadequate for identifying low-abundant transcripts and resolving complex populations of transcript isoforms. Direct long-read sequencing, while offering full-length information, suffers from throughput limitations, hindering the capture of low-abundance transcripts. To address these challenges, we introduce a targeted RNA enrichment strategy, rapid amplification of cDNA ends coupled with Nanopore sequencing (RACE-Nano-Seq). This method unravels the deep complexity of transcripts containing anchor sequences—specific regions of interest that might be exons of annotated genes, in silico predicted exons, or other sequences. RACE-Nano-Seq is based on inverse PCR with primers targeting these anchor regions to enrich the corresponding transcripts in both 5' and 3' directions. This method can be scaled for high-throughput transcriptome profiling by using multiplexing strategies. Through targeted RNA enrichment and full-length sequencing, RACE-Nano-Seq enables accurate and comprehensive profiling of low-abundance transcripts, often revealing complex transcript profiles at the targeted loci, both annotated and unannotated.
Visualization of the Evolution and Transmission of Circulating Vaccine-Derived Poliovirus (cVDPV) Outbreaks in the African Region
Since the creation of the Global Polio Eradication Initiative (GPEI) in 1988, significant progress has been made toward attaining a poliovirus-free world. This has resulted in the eradication of wild poliovirus (WPV) serotypes two (WPV2) and three (WPV3) and limited transmission of serotype one (WPV1) in Pakistan and Afghanistan. However, the increased emergence of circulating vaccine-derived poliovirus (cVDPV) and the continued circulation of WPV1, although limited to two countries, pose a continuous threat of international spread of poliovirus. These challenges highlight the need to further strengthen surveillance and outbreak responses, particularly in the African Region (AFRO). Phylogeographic visualization tools may provide insights into changes in poliovirus epidemiology, which can in turn guide the implementation of more strategic and effective supplementary immunization activities and improved outbreak response and surveillance. We created a comprehensive protocol for the phylogeographic analysis of polioviruses using Nextstrain, a powerful open-source tool for real-time interactive visualization of virus sequencing data. It is expected that this protocol will support poliovirus elimination strategies in AFRO and contribute significantly to global eradication strategies. These tools have been utilized for other pathogens of public health importance, for example, SARS-CoV-2, human influenza, Ebola, and Mpox, among others, through real-time tracking of pathogen evolution (https://nextstrain.org), harnessing the scientific and public health potential of pathogen genome data.
Cell Biology
Isolation of Podocyte Cell Fractions From Mouse Kidney Using Magnetic Activated Cell Sorting (MACS)
Glomerular diseases characterized by injury to post-mitotic epithelial cells called podocytes are a leading cause of chronic kidney disease. Yet, isolating podocytes from the kidney for transcriptomic, proteomic, and metabolomic studies has been a major technical challenge. Protocols utilizing glomerular sieving and laser capture methods are of limited use because they are not podocyte-specific but instead capture all four glomerular cell types. Here, we present a magnetic-activated cell sorting (MACS) method where podocytes are isolated from digested whole kidneys using antibodies specific to extracellular antigens on podocytes. Using microbeaded secondary antibodies binding to the podocyte-specific primary antibodies allows sorting of the podocytes using a magnet. This podocyte-only cell fraction is a unique source of in vivo–derived cells for molecular and cellular experiments.
Isolation of Mitochondria From Yeast to Estimate Mitochondrial Pools of Inorganic Phosphate
Mitochondria are dynamic organelles with essential roles in energetics and metabolism. Several metabolites are common to both the cytosolic and mitochondrial fractions of the cell. The compartmentalization of metabolites within the mitochondria allows specialized uses for mitochondrial metabolism. Inorganic phosphate (Pi) is one such critical metabolite required for ATP synthesis, via glycolysis and mitochondrial oxidative phosphorylation. Estimating total cellular Pi levels cannot distinguish the distribution of Pi pools across different cellular compartments, such as the cytosol and mitochondria, and therefore separate the contributions made toward glycolysis or other cytosolic metabolic processes vs. mitochondrial outputs. Quantifying Pi pools in mitochondria can therefore be very useful toward understanding mitochondrial metabolism and phosphate homeostasis. Here, we describe a protocol for the fairly rapid, efficient isolation of mitochondria from Saccharomyces cerevisiae by immunoprecipitation for quantitative estimation of mitochondrial and cytosolic Pi pools. This method utilizes magnetic beads to capture FLAG-tagged mitochondria (Tom20-FLAG) from homogenized cell lysates. This method provides a valuable tool to investigate changes in mitochondrial phosphate dynamics. Additionally, this protocol can be coupled with LC–MS approaches to quantitatively estimate mitochondrial metabolites and proteins and can be similarly used to assess other metabolite pools that are partitioned between the cytosol and mitochondria.
Immunology
Evaluation of In Vitro Cytotoxic Activity of CAR-T Cells Using Patient-Derived Organoids
Adoptive immune cell therapy, especially chimeric antigen receptor T (CAR-T) cells, has emerged as a promising strategy in solid tumor treatment, owing to its unique ability to specifically recognize and effectively eliminate tumor cells. Patient-derived organoids (PDOs) offer a robust and physiologically relevant platform for assessing the safety and efficacy of CAR-T-cell-based therapies. We now describe a detailed protocol for an in vitro evaluation system based on the co-culture of PDOs and CAR-T cells. This system encompasses the establishment of tumor organoids from patient tumor specimens, the isolation of T cells from matched peripheral blood mononuclear cells (PBMCs), and the generation of antigen-specific CAR-T cells. Through the use of fluorescent labeling to visualize different cells and apoptosis-related events post-interaction, along with quantitative analyses of T-cell proliferation, tumor organoid apoptosis, and the secretion of immune effector molecules, this system enables a robust and multifaceted evaluation of CAR-T cell cytotoxicity in vitro. Collectively, this co-culture system provides a systematic and reproducible in vitro platform for evaluating the functional activity of CAR-T cells and advancing research in tumor immunology and immunotherapy.
Microbiology
Untargeted Metabolomics of Epimastigote Forms of Trypanosoma cruzi
Trypanosoma cruzi, the causative agent of Chagas disease, faces significant metabolic challenges due to fluctuating nutrient availability and oxidative stress within its insect vector. Metabolomic techniques, such as gas chromatography–mass spectrometry (GC–MS), have been widely used to study the adaptive mechanisms of the parasite. This article describes a standardized method for the untargeted metabolomics analysis of T. cruzi epimastigote, covering parasite cultivation, sample deproteinization with methanol, metabolite extraction, derivatization with BSTFA, and GC–MS analysis. To ensure robustness and reproducibility, statistical analysis uses univariate tests, as well as multivariate approaches such as principal component analysis (PCA) and partial least squares (PLS) regression. The protocol offers a reliable and sensitive method to study metabolic responses in T. cruzi under environmental stress, with low biological variability and high reproducibility.
Rapid and Multiplex Diagnosis of Malaria Using Chelex-100 Extraction and LAMP-MS Assay
Malaria remains a major public health threat, especially in tropical and subtropical regions. Accurate and rapid diagnosis is essential for effective disease management and control, yet conventional malaria diagnostics, including blood smear microscopy using Giemsa staining, PCR, and rapid diagnostic tests (RDTs), are limited by the need for trained personnel, reliance on laboratory infrastructure, and reduced sensitivity at low parasite densities, respectively. This protocol details an innovative, rapid, and economical diagnostic platform combining a simplified Chelex-100 resin-based nucleic acid extraction method with a multiplex loop-mediated isothermal amplification microscanner (LAMP-MS) assay. The malaria diagnostic platform enables simultaneous detection of Plasmodium falciparum (Pf), Plasmodium vivax (Pv), pan-malaria (Pan), and an internal control (IC) within 40 min, from DNA extraction to result interpretation. It demonstrates sensitivity and specificity comparable to traditional PCR-based diagnostics, making it a practical and scalable solution for use in resource-constrained environments.
Molecular Biology
Protocol for Generation of Single-Gene Knockout in Hard-to-Transfect THP1 Cell Lines Using CRISPR/Cas9
This protocol provides a step-by-step approach for generating single-gene knockout in hard-to-transfect suspension immune cell lines like THP1, specifically demonstrated by knocking out the GSDMD gene. By employing CRISPR-Cas9 system delivered via lentivirus, this protocol enables precise gene disruption through targeted single-guide RNAs (sgRNAs). Key steps include designing specific sgRNAs, cloning them into a CRISPR vector, viral packaging, and transducing the target cells, followed by selection and validation. This optimized protocol is particularly useful for functional studies in immune cells, allowing researchers to reliably explore gene function in complex cellular pathways.
APEX2 RNA Proximity Labeling in Mammalian Cell Lines With Low Biotin Permeability
The subcellular localization of RNA plays a critical role in various biological processes, including development and stress response. Proximity labeling eases the detection of localized transcripts and protein enrichment compared to previous techniques that rely on biochemical isolation of subcellular structures. The rapid reaction and small labeling radius of APEX2 make it an attractive alternative to other proximity labeling approaches, such as BioID. However, we found that standard protocols for APEX proximity labeling fail in human induced pluripotent stem cells. Moreover, standard protocols yield heterogeneous labeling of biomolecules across single cells in MCF10A breast epithelial cells. Our results indicate that low biotin permeability in these cell lines is the main cause for failed or inefficient labeling. This protocol outlines improved labeling by combining the rapid hydrogen peroxide-driven APEX2 reaction with the addition of a mild detergent during biotin incubation. This adaptation leads to efficient proximity labeling in hiPSCs and more homogeneous biotinylation across single cells in MCF10As. The adapted protocol extends the use of APEX2 proximity labeling to cell lines with poor biotin permeability.
Assessing the Efficiency of Double-Strand Break Repair Mediated by Homologous Recombination and Non-homologous End-Joining Pathways in Saccharomyces cerevisiae
The DNA double-strand breaks (DSBs) generated by exogenous and endogenous factors are repaired by two pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). These two pathways compete for DSB repair, and the choice of pathway depends on the context of the DNA lesion, the stage of the cell cycle, and the ploidy in the yeast Saccharomyces cerevisiae. However, the mechanistic details of the DSB repair pathway choice and its consequences for S. cerevisiae genome stability remain unclear. Here, we present PCR-based and cell-based assays as well as data analysis methods to quantitatively measure the efficiency of HR and NHEJ at DSBs in S. cerevisiae. An intermolecular recombination assay between plasmid and chromosomal DNA involving G-quadruplex DNA and a “suicide-deletion” assay have been utilized to evaluate the efficiency of HR and NHEJ, respectively. These streamlined protocols and optimized growth conditions can be used to identify the NHEJ- and HR-deficient S. cerevisiae mutant strains.
Neuroscience
Two-photon (2P) Microscopy to Study Ca2+ Signaling in Astrocytes From Acute Brain Slices
Since the discovery that astrocytes are characterized by Ca2+-based excitability, investigating the function of these glial cells within the brain requires Ca2+ imaging approaches. The technical evolution from chemical fluorescent Ca2+ probes with low cellular specificity to genetically encoded indicators (GECIs) has enabled detailed analysis of the spatial and temporal features of intracellular Ca2+ signal. Different imaging methodologies allow the extraction of distinct information on calcium signals in astrocytes from brain slices, with resolution ranging from cell populations to single cells up to subcellular domains.Here, we describe 2-photon laser scanning microscopy (2PLSM) Ca2+ imaging in astrocytes from the somatosensory cortex (SSCx) of adult mice in ex vivo acute cortical slices, performed using two genetically encoded Ca2+ indicators, i.e., cytosolic GCaMP6f and endoplasmic reticulum-targeted G-CEPIA1er. The main advantage of the 2PLSM technique, compared to single-photon microscopy, is the possibility to go deeper in the tissue while avoiding photodamage, by limiting laser excitation to a single focal plane. The fluorescent signal of the indicator is analyzed offline in different compartments—soma, proximal processes, and microdomains—for GCaMP6f experiments and in the perinuclear, somatic area for G-CEPIA1er. The analysis of Ca2+ signal from different compartments, although not providing a value of absolute concentration, allows a critical comparison of the degree of astrocyte activation between different experimental conditions or mouse models. Moreover, the analysis of G-CEPIA1er signal, which reveals metabotropic receptor activation as a dynamic decrease in free Ca2+ in the endoplasmic reticulum (ER), can provide information on possible alterations in this critical second messenger pathway in astrocytes, including, for example, steady-state ER Ca2+ levels and kinetics of Ca2+ release.
An Alternative Gene Editing Strategy Using a Single AAV Vector
We recently developed an approach for cell type–specific CRISPR/Cas9 editing and transgene expression using a single viral vector. Here, we present a protocol describing how to design and generate plasmids and adeno-associated viruses (AAVs) compatible with this single-vector gene editing approach. This protocol has four components: (1) guide RNA (gRNA) design to target specific genes of interest, (2) ligation and cloning of CRISPR-competent AAV vectors, (3) production of vector-containing AAVs, and (4) viral titer quantification. The resultant vectors are compatible for use with mouse lines expressing the Cas9 protein from Streptococcus pyogenes (SpCas9) and Cre recombinase to enable selective co-expression of standard neuroscience tools in edited cells. This protocol can produce AAVs of any serotype, and the resulting AAVs can be used in the central and peripheral nervous systems. This flexible approach could help identify and test the function of novel genes affecting synaptic transmission, circuit activity, or morphology with a single viral injection.
Live Cell Imaging to Monitor Axonal Pruning in Drosophila Motor Neurons
Over the lifespan of an individual, brain function requires adjustments in response to environmental changes and learning experiences. During early development, neurons overproduce neurite branches, and neuronal pruning removes the unnecessary neurite branches to make a more accurate neural circuit. Drosophila motoneurons prune their intermediate axon bundles rather than the terminal neuromuscular junction (NMJ) by degeneration, which provides a unique advantage for studying axon pruning. The pruning process of motor axon bundles can be directly analyzed by real-time imaging, and this protocol provides a straightforward method for monitoring the developmental process of Drosophila motor neurons using live cell imaging.
Construction of Large Cranial Windows With Nanosheet and Light-Curable Resin for Long-term Two-Photon Imaging in Mice
In vivo two-photon imaging of the mouse brain is essential for understanding brain function in relation to neural structure; however, its application is limited by the size and mechanical stability of conventional cranial windows. Here, we present the procedure of a large-scale cranial window technique based on the nanosheet incorporated into light-curable resin (NIRE) method. This approach utilizes a biocompatible polyethylene-oxide-coated CYTOP (PEO-CYTOP) nanosheet combined with light-curable resin, allowing the window to conform to the brain’s curved surface. The protocol enables long-term, high-resolution, and multiscale imaging—from subcellular structures to large neuronal populations—in awake mice over several months.
Quantification of Neural Progenitor Cells From Zika Virus-Infected Zebrafish Embryos
Zika virus (ZIKV), an arthropod-borne orthoflavivirus, has emerged as a global health concern due to its ability to cause severe fetal neurological disorders, leading to the congenital Zika syndrome (CZS) in neonates. Vertical transmission during pregnancy can alter neural progenitor cell (NPC) proliferation and differentiation and induce apoptosis, leading to microcephaly and other neurodevelopmental abnormalities. While mammalian models have been used to study the impact of ZIKV on NPC behavior, limitations such as high costs, dedicated time, and ethical constraints have fostered the exploration of alternative systems. The zebrafish embryo constitutes an advantageous in vivo model for studying ZIKV neuropathogenesis. Indeed, ZIKV infection phenocopies several features of the CZS while sharing a conserved neuroanatomical layout and offering genetic plasticity and unique accessibility to the infected brain compared to mammals. Here, we describe a protocol for characterizing ZIKV-induced defects of NPCs in this zebrafish model, relying on whole animal flow cytometry.
Derivation and Culture of Enriched Phrenic-Like Motor Neurons From Human iPSCs
The fatal motor neuron (MN) disease amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of the phrenic MNs (phMNs) controlling the activity of the diaphragm, leading to death by respiratory failure. Human experimental models to study phMNs are lacking, hindering the understanding of the mechanisms of phMN degeneration in ALS. Here, we describe a protocol to derive phrenic-like MNs from human induced pluripotent stem cells (hiPSC-phMNs) within 30 days. During spinal cord development, phMNs emerge from specific MN progenitors located in the dorsalmost MN progenitor (pMN) domain at cervical levels, under the control of a ventral-to-dorsal gradient of Sonic hedgehog (SHH) signaling and a rostro-caudal gradient of retinoic acid (RA). The method presented here uses optimized concentrations of RA and the SHH agonist purmorphamine, followed by fluorescence-activated cell sorting (FACS) of the resulting MN progenitor cells (MNPCs) based on a cell-surface protein (IGDCC3) enriched in hiPSC-phMNs. The resulting cultures are highly enriched in MNs expressing typical phMN markers. This protocol enables the generation of hiPSC-phMNs and is highly reproducible using several hiPSC lines, offering a disease-relevant system to study mechanisms of respiratory MN dysfunction. While the protocol has been validated in the context of ALS research, it can be adopted to study human phrenic MNs in other research fields where these neurons are of interest.