STAR Protocols

Protocol for pooled FACS-based CRISPR knockout screening in human iPSC-derived microglia Here, we present a protocol for CRISPR knockout screening in human induced pluripotent stem cell (hiPSC)-derived microglia (iMGL) using lentiviral delivery of CRISPR-Cas9 and co-transduction of VPX virus-like particles (VPX-VLPs). We first describe large-scale production of iMGL from hiPSCs, production of the lentiviral and VPX-VLP libraries, and titration. Next, we describe how to perform a pooled CRISPR screen for phagocytosis including the computational analysis pipeli...

Protocol for pooled FACS-based CRISPR knockout screening in human iPSC-derived microglia #protocol #starprotocols #cellpress

Quantification and analysis of multiplexed fluorescence in situ hybridization data using open-source tools Here, we present a protocol to quantify and analyze multiplexed fluorescence in situ hybridization (mFISH) data using two open-source tools, FijiFISH and RUHi. FijiFISH, an ImageJ-based plugin, enables image registration, cell segmentation, and gene expression quantification. RUHi, an R-based package, supports dimensionality reduction, clustering, and visualization through both code and a Shiny app. The protocol also accommodates experimentally induced exogenous fluorophores, providing multimoda...

Quantification and analysis of multiplexed fluorescence in situ hybridization data using open-source tools #protocol #starprotocols #cellpress

I am pleased to serve as one of the Guest Editors for the upcoming @cp-starprotocols.bsky.social focused issue on “Protocols in Metabolomics and Lipidomics Research.”

We welcome detailed and comprehensive protocols that provide broad utility to the community.

#Metabolomics #Lipidomics #MS #NMR

Maddie, Andoni and Agustin, the three PhD students in my lab just had their paper accepted in @cp-starprotocols.bsky.social!! 🥳 They describe a protocol for quantifying the horizontal and vertical distribution of junctional proteins in confluent, fixed epithelial cells. doi.org/10.1016/j.xp...

Cell Press: STAR Protocols STAR Protocols is an open access, peer-reviewed journal from Cell Press. We offer structured, transparent, accessible, and repeatable step-by-step experimental and computational protocols from all are...

🚀 Thrilled to kick off my Bluesky feed with some exciting science!

Are you (or your colleagues) working with ex vivo tissue culture or tissue infection models? 🧫🦠
We just published a new protocol in @cp-starprotocols.bsky.social 🎉

star-protocols.cell.com/protocols/4475

Protocol to culture cells on genipin-mixed collagen gels with different stiffnesses The stiffness of extracellular matrices is critical for cellular functions such as growth and differentiation. Here, we present a protocol for culturing cells on stiffness-modulated collagen gels by adding genipin, an amine crosslinker with low cytotoxicity. We describe the steps for preparing collagen gels with genipin, culturing the cells, and immunofluorescent staining of the cells on the gels. This protocol has potential applications in the analysis of function or protein/gene expression in ...

Protocol to culture cells on genipin-mixed collagen gels with different stiffnesses #protocol #starprotocols #cellpress

Protocol for designing and developing a population-based individualized intervention planning assistant for overweight and obesity Overweight and obesity are multifactorial health problems requiring innovative analytical approaches. We present a protocol to develop a population-based individualized intervention planning assistant (PIIPA) using supervised machine learning, Shapley additive explanations (SHAP), and a long short-term memory (LSTM) model. We describe steps for constructing interpretable machine learning (ML) models, identifying key predictors, and simulating BMI trajectories under interventions. We then detail ...

Protocol for designing and developing a population-based individualized intervention planning assistant for overweight and obesity #protocol #starprotocols #cellpress

Protocol to predict the origin of cancer of unknown primary from gene expression data using BPformer Patients diagnosed with cancer of unknown primary (CUP) have poor prognoses, highlighting the need to identify primary sites for targeted therapeutic intervention. Here, we present a protocol to predict the primary sites of CUP based on gene expression data using BPformer. We describe steps for installing software, collecting and preprocessing data, and training the BPformer model. We then detail procedures on using BPformer via a visualized web server. For complete details on the use and execut...

Protocol to predict the origin of cancer of unknown primary from gene expression data using BPformer #protocol #starprotocols #cellpress

Protocol for experimental infections with viruses in mosquitoes using a glass artificial feeder Here, we present a protocol for infecting Aedes aegypti mosquitoes with Zika virus (ZIKV) (Flaviviridae) using a glass artificial feeder that mimics natural feeding conditions. We describe steps for preparing adult mosquitoes, assembling the feeder, mixing the infectious blood meal, and conducting infection experiments. The feeder incorporates a bovine gut membrane and a thermostatic circulator to maintain the blood meal at 37°C, ensuring consistent feeding and reproducible infection outcomes. F...

Protocol for experimental infections with viruses in mosquitoes using a glass artificial feeder #protocol #starprotocols #cellpress

Protocol for reproducible EZ clearing and labeling, including optimized steps and quantified fluorescence retention on mouse tissue Here, we present a protocol for preserving fluorescent markers using tissue clearing and immunolabeling in mouse tissue such as sciatic nerve, gut, and spinal cord. We describe steps for maintaining tetrahydrofuran (THF) under an inert atmosphere, EZ clearing, immunolabeling, imaging with custom lightsheet microscopy setups, and post-processing. This protocol was refined to ensure stable and reliable clearing across various tissue types. Key improvements include inert atmosphere THF handling, ti...

Protocol for reproducible EZ clearing and labeling, including optimized steps and quantified fluorescence retention on mouse tissue #protocol #starprotocols #cellpress

Protocol for contactless and instantaneous line-current data exchange between MATLAB and a drone-deployable sensor on overhead transmission lines Here, we present a protocol for fabricating a drone-deployable electric current sensor for real-time, remote monitoring of electric currents in overhead transmission lines. We describe steps for designing and manufacturing the board in Autodesk EAGLE and the signal processing units. We then detail procedures for programming routines on the Arduino Due to process data and curve visualization environment for Bluetooth-received data in MATLAB. The system supports data exchange up to 60 meters, cont...

Protocol for contactless and instantaneous line-current data exchange between MATLAB and a drone-deployable sensor on overhead transmission lines #protocol #starprotocols #cellpress

Protocol for trapping transient endogenous formaldehyde in live cells to visualize its signaling Formaldehyde is emerging as a key signaling modulator. Here, we present Trami, a protocol that traps endogenous formaldehyde in live cells and converts its transient signals into stable proximal protein labels for subsequent signaling study. We describe probe design, live-cell administration, and imaging procedures for signaling deconvolution. Trami offers a powerful tool for elucidating formaldehyde-mediated signaling pathways. For complete details on the use and execution of this protocol, ple...

Protocol for trapping transient endogenous formaldehyde in live cells to visualize its signaling #protocol #starprotocols #cellpress

Protocol for stromal vascular fraction isolation from inguinal subcutaneous white adipose tissue and beige adipocyte differentiation Research on inducing beige adipocytes to combat obesity and its comorbidities is growing, with stromal vascular fraction (SVF) from white adipose tissue showing potential to differentiate into both white and beige adipocytes. Here, we present a protocol for isolating the SVF from the iWAT (inguinal white adipose tissue) of mice and differentiating them into mature beige adipocytes. This protocol can be used to analyze the translational regulation and metabolic processes of beige adipocytes in vi...

Protocol for stromal vascular fraction isolation from inguinal subcutaneous white adipose tissue and beige adipocyte differentiation #protocol #starprotocols #cellpress

Protocol to detect dilution cycles in chemostat experiments and estimate growth rate slopes with linear modeling with R software chemostat_regression Chemostat growth chambers measure optical density over time and require manual calculation of growth rates. Here, we present chemostat_regression, R software that enables users to automatically identify chemostat cycles and estimate growth rate using a linear regression approach. We describe steps for creating requisite software environment(s), formatting input data, executing the software via command line/RStudio/R-Shiny, interpreting results, assessing the validity of results, and modifying in...

Protocol to detect dilution cycles in chemostat experiments and estimate growth rate slopes with linear modeling with R software chemostat_regression #protocol #starprotocols #cellpress

Protocol for immunofluorescence imaging of epitope-tagged protein in Arabidopsis shoot apical meristem Proteins expressed in or translocated to the shoot apical meristem (SAM) often play pivotal roles in regulating plant development. Here, we present a step-by-step protocol for immunofluorescence imaging to analyze epitope (FLAG)-tagged proteins in the Arabidopsis SAM. This technique may be suitable to visualize mobile proteins because small epitope tags are less disruptive than a relatively large fluorescent protein fusion. For complete details on the use and execution of this protocol, please r...

Protocol for immunofluorescence imaging of epitope-tagged protein in Arabidopsis shoot apical meristem #protocol #starprotocols #cellpress

Protocol for multiplex whole-mount RNA fluorescence in situ hybridization combined with immunohistochemistry in the mosquito brain Advances in sequencing technologies have enabled transcriptional profiling of previously understudied yet critical species, such as mosquitoes. Here, we present a protocol for multiplex whole-mount RNA fluorescence in situ hybridization combined with immunohistochemistry in the Anopheles gambiae brain. We describe steps for dissection, fixation, probe hybridization, hybridization chain reaction (HCR) probe detection, and primary antibody application. We then detail procedures for tissue preparat...

Protocol for multiplex whole-mount RNA fluorescence in situ hybridization combined with immunohistochemistry in the mosquito brain #protocol #starprotocols #cellpress

Protocol for simplified parallel perturbations using an abridged long non-coding RNA CRISPR library High-throughput CRISPR interference (CRISPRi) screens are invaluable for discovering novel functional genes, but applying such screens to long non-coding RNAs (lncRNAs) is more challenging. Here, we present a protocol for designing and executing pooled CRISPRi screens targeting lncRNAs using an abridged cell-type-specific dual single-guide RNA (sgRNA) library. We describe steps for library design and synthesis, followed by stable lentiviral transduction. We then provide guidelines for performing...

Protocol for simplified parallel perturbations using an abridged long non-coding RNA CRISPR library #protocol #starprotocols #cellpress

Protocol for integrating immunohistochemistry and H&E annotations with Xenium data at single-cell resolution Targeted investigation of spatial transcriptomic data benefits from versatile integration of customized external information. Here, we present a protocol for embedding irregular field-of-view annotations and immunohistochemistry data into a Xenium dataset at the single-cell resolution using open-source tools. We detail steps for image registration and demonstrate how to integrate spatially aligned information into a Scanpy-compatible anndata object using a head and neck squamous cell carcinoma s...

Protocol for integrating immunohistochemistry and H&E annotations with Xenium data at single-cell resolution #protocol #starprotocols #cellpress

Protocol for quantifying PARP inhibitor-induced changes in PARP1 dynamics and activity through live-cell imaging Poly(ADP-ribose) polymerase 1 (PARP1) is a crucial DNA repair protein and a target of PARP inhibitors (PARPi), which suppress its catalytic activity while also altering its association with damaged chromatin. Here, we present a live-cell imaging protocol for obtaining high-quality kinetics of fluorescently labeled PARP1 and poly(ADP-ribose) (PAR)-binding proteins at micro-irradiation-induced DNA damage sites in untreated and PARPi-treated cells. This approach can be easily adapted to other DNA r...

Protocol for quantifying PARP inhibitor-induced changes in PARP1 dynamics and activity through live-cell imaging #protocol #starprotocols #cellpress

Protocol for selective DREADD-based chemogenetic inhibition of GABAergic amygdala neurons receiving hippocampal projections in rats Designer receptors exclusively activated by designer drugs (DREADDs) are a highly precise and effective method for manipulating neurons and other excitable cells. Here, we present an approach that combines viral anterograde transsynaptic transport with Gαi/o DREADD neuronal inhibition to control targeted neurons in a rat model. We describe how to implement this technique, from surgical the procedure to the behavioral assay. This protocol enables researchers to manipulate target neurons receiving...

Protocol for selective DREADD-based chemogenetic inhibition of GABAergic amygdala neurons receiving hippocampal projections in rats #protocol #starprotocols #cellpress

Protocol to set up the CellMinerCDB pharmacogenomics analysis web application with custom cell line data CellMiner Cross-Database (CellMinerCDB) is an interactive web application for integrating and analyzing molecular and pharmacological data across human cancer cell lines. Here, we detail the setup process, including installing necessary software, preparing compatible datasets, and customizing configuration files; we use sarcoma data as an example. The protocol involves data loading, software configuration, and deployment to enable univariate and multivariate analyses. For complete details on the...

Protocol to set up the CellMinerCDB pharmacogenomics analysis web application with custom cell line data #protocol #starprotocols #cellpress

Protocol to set up the immuno-microfluidic system for rapid measurement of salivary cortisol level Microfluidics has versatile applications in the field of biosensors. We present a protocol for assembling a microfluidic system to rapidly measure cortisol in saliva. This protocol prepares microfibrous reactors immobilized with antibodies as an alternative to a 96-well plate and integrates a flow system, reactor platform, and detection device. The output current signal directly reflects cortisol levels for rapid evaluation. For complete details on the use and execution of this protocol, please ...

Protocol to set up the immuno-microfluidic system for rapid measurement of salivary cortisol level #protocol #starprotocols #cellpress

Protocol for quantifying extracellular and intracellular ATP from macrophages upon inflammasome activation using a luciferin-luciferase technique Extracellular ATP triggers several cellular responses, while intracellular ATP depletion primes cells for death. Here, we present a protocol for the quantification of extracellular and intracellular ATP from bone marrow-derived macrophages upon inflammasome activation using a luciferin-luciferase technique. We describe steps for macrophage isolation, differentiation, and inflammasome activation with real-time ATP measurement. The protocol allows quantitative determination of intracellular ATP an...

Protocol for quantifying extracellular and intracellular ATP from macrophages upon inflammasome activation using a luciferin-luciferase technique #protocol #starprotocols #cellpress

Protocol to assess the cytotoxicity of autologous human skin immune cells against senescent fibroblasts Immune clearance of senescent cells contributes to their control in aging tissues; however, it remains unclear how immunity against senescent cells is regulated in humans. Here, we provide a protocol to measure the cytotoxicity of autologous human skin immune cells against senescent dermal fibroblasts. We describe the ex vivo co-culture experiments using normal and senescent fibroblasts together with autologous immune cells isolated from the human skin. This protocol is applicable to understandi...

Protocol to assess the cytotoxicity of autologous human skin immune cells against senescent fibroblasts #protocol #starprotocols #cellpress

Protocol for monitoring the growth of Solanum betaceum non-embryogenic callus using electrochemical impedance spectroscopy Callus cultures have important biotechnological applications. Here, we describe the use of electrochemical impedance spectroscopy (EIS) as a real-time and non-destructive approach to monitor callus growth. We describe the steps to obtain in vitro tamarillo clones from seeds and from these non-embryogenic calluses. We detail the steps for the preparation of the EIS apparatus and experiment and a parallel growth assay. We also provide complementary microscopy assays to complement the EIS experimen...

Protocol for monitoring the growth of Solanum betaceum non-embryogenic callus using electrochemical impedance spectroscopy #protocol #starprotocols #cellpress

Protocol for Hi-C-based identification of chromosomal inversions in mosquitoes Chromosomal inversions play an important role in the genomic evolution and adaptation of mosquito species. Here, we present a protocol for detecting chromosomal inversions in mosquitoes using Hi-C technology and chromatin contact heatmaps. We describe the steps for Hi-C library preparation using the Hi-C Arima+ kit, along with the Arima Library Prep Module, and provide several adaptations for mosquito samples. We then outline procedures for Hi-C data analysis, Hi-C heatmap generation, and the id...

Protocol for Hi-C-based identification of chromosomal inversions in mosquitoes #protocol #starprotocols #cellpress

Protocol for single-base quantification of RNA m5C by pyrosequencing 5-methylcytosine (m5C) is a common RNA modification found in both coding and non-coding RNAs. Here, we present a protocol for RNA-m5C-pyroseq for quantitative, single-nucleotide resolution analysis of RNA methylation. We describe steps for designing primers for the target region of RNA, performing bisulfite conversion of RNA, and using pyrosequencing to measure RNA methylation in cytosine. We then detail procedures for all the steps, including RNA isolation, bisulfite conversion, cDNA synthesis,...

Protocol for single-base quantification of RNA m5C by pyrosequencing #protocol #starprotocols #cellpress

Protocol for isolating and characterizing extracellular vesicles by ultracentrifugation from bone marrow-derived macrophages Extracellular vesicles (EVs) enable the transmission of crucial molecular components between the parental and recipient cells. Macrophages can polarize into two distinct macrophage phenotypes, thereby exerting diverse effects on recipient cells. Here, we present a protocol for the direct isolation of EVs from bone marrow-derived macrophages (BMDMs) using ultracentrifugation. We describe steps for culturing BMDMs and macrophage polarization. We then detail procedures for further characterizing th...

Protocol for isolating and characterizing extracellular vesicles by ultracentrifugation from bone marrow-derived macrophages #protocol #starprotocols #cellpress

Protocol for a human placental explant model to study acute responses to pathogens Here, we present a protocol for studying infections caused by pathogens associated with intrauterine complications—Listeria monocytogenes, Plasmodium falciparum, and Toxoplasma gondii—using an ex vivo placental explant model. We describe procedures for pathogen culture, preparation of placental explants using an air-liquid interface system, and infection assays. We detail procedures for tissue dissociation and CD45+ cell enrichment. This protocol allows short-term placental infection studies. Fo...

Protocol for a human placental explant model to study acute responses to pathogens #protocol #starprotocols #cellpress

Protocol for contactless electric current sensing, processing, and storage using a drone-integrable sensor Here, we present a protocol for implementing a contactless, drone-mounted current sensor to measure and record electric current norms in overhead transmission lines. We describe steps for designing, fabricating, and assembling the printed circuit board (PCB) and programming the Arduino Mega 2560 as the data processor. We further outline the integration of MATLAB scripts for graphical visualization of the sensed currents, ensuring efficient data interpretation. This protocol enables accurate labo...

Protocol for contactless electric current sensing, processing, and storage using a drone-integrable sensor #protocol #starprotocols #cellpress