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...

A non-invasive system can measure nutrient uptake and photosynthate partitioning simultaneously while allowing organisms to metabolize without interference in the process of measurement. Here, we present a protocol for detecting nutrient uptake and photosynthate partitioning in rice seedling using a non-invasive system. We describe steps for preparing plant material and material for the non-invasive system. We then detail procedures for setting up and applying the non-invasive measurement system...

Here, we present a protocol to study neural circuits between the paraventricular thalamus (PVA) and the bed nucleus of the stria terminalis (BNST) in mice by combining calcium imaging with optogenetic stimulation of axon terminals. We describe steps for delivering GCaMP6f and ChrimsonR viruses and implanting the gradient-index (GRIN) lens for use with an Inscopix microscope. We then detail procedures for single-neuron tracking over an extended period through longitudinal recording and data smoot...

Vertical-looking radars (VLRs) detect individual flying animals up to ∼2 km above ground and characterize their flight track, timing, wing movement, size, and shape. We present a protocol for calculating individual vertical movement characteristics and producing diel vertical movement profiles using the BirdScan MR1 VLR, based on flight altitude detection and timing. The protocol includes steps for data preparation and analysis. This protocol promotes a more detailed understanding of migration e...

The human placental explant model preserves the placenta’s natural structure, enabling studies on whole-tissue response to exposures or growth conditions. It outlines procedures for placental collection, isolation, and culturing, followed by exposure to conditions of interest. Additionally, it details sample collection of explant tissue and culture supernatant after exposure for downstream assessments of placental function. This protocol provides a framework for evaluating ex vivo responses of h...

Dormant disseminated cancer cells are responsible for late relapses of breast cancer. Here, we present a protocol to orthotopically inject 4T07-mCherry breast cancer cells into mice and describe how to resect the primary tumor. We detail steps for collecting and processing the lungs to detect and quantify cancer cells using an algorithm-based approach. This protocol allows studying the induction, maintenance, and awakening from metastatic dormancy. For complete details on the use and execution o...

Human cerebral organoids (hCOs) provide an excellent model for the study of human brain development and disease. Here, we present a protocol to obtain hCOs directly from two-dimensional (2D) pluripotent stem cell (PSC) cultures, avoiding cell dissociation…

Understanding cortical microstructure related to timing is key to studying aging and neurodegenerative diseases. Here, we present a protocol for identifying brain regions involved in time perception using microstructural measures from multi-shell diffusion MRI (dMRI). We outline steps for processing data, applying NODDI (neurite orientation dispersion and density imaging) modeling to extract cortical neurite parameters, and quantifying neurite microstructure patterns. Finally, we detail procedur...

Vitreous infiltrating immune cells represent an accessible tissue sample that can provide valuable information on the intraocular immune response without compromising the structural and functional integrity of the eye. Here, we present our customized protocol for isolating and characterizing vitreous immune cell infiltrates by flow cytometry and single-cell transcriptomics. The protocol includes surgical modifications for sample collection and other measures to maximize the yield of immune cells...

Analysis of metabolites provides key insights into brain physiology and function. Due to post-mortem metabolism, both the euthanasia method and dissection time can make a critical difference. Here, we describe a protocol to euthanize rodents by microwave irradiation. This workflow details steps for animal placement, tissue fixation, and post-fixation processing. This protocol enables the rapid halting of metabolic activity for the accurate assessment of the metabolome in situ for analyses such a...

We present a protocol for predicting γ-tocotrienol (γT3) and δ-tocotrienol (δT3) binding to colorectal cancer-related proteins using Schrödinger’s Maestro and Glide. The protocol describes protein preparation, ligand processing, and receptor grid generation, followed by docking simulations. It outputs binding affinities (kcal/mol) and interaction maps, with δT3 expected to bind slightly more strongly. This workflow includes checkpoints and troubleshooting, providing a reproducible framework suit...

Here, we present a step-by step protocol to visualize intracellular aggregations in Arabidopsis mutants with cell wall secretion defects using FM4-64, a lipophilic styryl dye. We describe steps for growing seedlings, staining them with FM4-64, and…

Alternating current–direct current (AC-DC) electropenetrography (EPG) is a non-invasive approach for quantifying parasitic arthropods’ probing and ingestion behaviors inside host tissues. Here, we present a procedure for using the Observer XT behavioral coding software to synchronize EPG waveforms with video recordings. We describe the steps for performing, importing, and analyzing simultaneous recordings. This protocol has potential applications for the initial correlation of waveforms with art...

Ti2Bi2C is a ternary atomically layered carbide MAX phase with potential applications in radiation shielding under elevated temperature conditions. Here, we present a protocol for synthesizing the double-A-layer MAX phase Ti2Bi2C from elemental Ti, Bi, and C powders. The approach involves steps for powder mixing and homogenization, pressing, vacuum sealing in quartz ampules, and high-temperature solid-state reaction at 1,000°C for 48 h. Powder X-ray diffraction confirms Ti2Bi2C formation as the ...

Here, we present a protocol for identifying interactors of circular RNAs (circRNAs), specifically circDlc1(2), in the mouse cortex. We outline steps for tissue dissociation and UV crosslinking to maintain native interactions, followed by an RNA pull-down to isolate the circRNA and its associated molecules. The protocol has been optimized to detect potential protein interactors and can be adapted for use in other regions of the mouse nervous system. For complete details on the use and execution o...

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...

Schirmer strips are widely regarded as the gold standard for tear fluid collection. However, their use presents several challenges for proteomic analysis. Here, we present a protocol for extracting tear proteins from Schirmer strips. We describe steps for acquisition and handling of strips, extraction buffer preparation, strip preparation, and protein extraction. This protocol is designed to improve protein yield and facilitate proteomic workflows and is adaptable for various protein-based studi...

Activatable photoacoustic imaging probes offer a strategy to efficiently reduce background noise from endogenous chromophores. We present a protocol for tumor imaging in mice using an activatable covalent photoacoustic imaging probe, NOx-JS013. We describe steps for synthesizing NOx-JS013, in vitro and in situ validation through gel-based activity-based protein profiling and cellular imaging, and tumor imaging of aggressive prostate cancer mouse models. This approach provides a strategy for miti...

Traditionally, midbody remnants (MBRs) are isolated from cell culture medium using ultracentrifugation, which is expensive and time consuming. Here, w…

Drosophila wing and eye-antennal imaginal discs are excellent models for studying tissue development and cell behavior. Here, we present a protocol for long-term cultivation and imaging of wing and eye-antennal imaginal discs. We describe the mounting, dissection, and ex vivo cultivation of imaginal discs. We further detail 4D time-lapse image acquisition with minimized phototoxicity and image restoration using a machine-learning-based algorithm. This protocol is suitable for studying dynamic ce...

Investigating proteases and protease inhibitors produced and secreted by primary cells is complicated by the presence of exogenous ones in cell culture medium containing fetal bovine serum. With this protocol, we generate human monocyte-derived macrophages in the absence of exogenous proteases and protease inhibitors by using a serum replacement for the final 24 h of macrophage culture. We describe steps for seeding and differentiating monocytes into macrophages. We then detail procedures for ma...

Identification and functional characterization of candidate effector proteins of pathogens are essential for understanding plant-microbe interactions. Here, we present a protocol for probing candidate effector activity using the Prf-based plant cell death phenotype. We describe steps for co-infiltrating plant leaves with a Prf protein and the effector and visually assess the cell death phenotype as a readout for detecting effector activity. The protocol enables the screening of candidate effecto...

Arrestins associate with phosphorylated G protein-coupled receptors and undergo conformational changes. Here, we present a protocol for measuring β-arrestin1-phosphorylated peptide interaction and analyzing conformational dynamics of β-arrestin1. We describe steps for constructing expression plasmids, expressing and purifying β-arrestin1, and performing hydrogen/deuterium exchange mass spectrometry analysis of V2Rpp-β-arrestin1 complex. We then detail procedures for measuring binding affinity us...