Protocol for preparing metabolically reprogrammed human CAR T cells and evaluating their in vitro effects.

Chimeric antigen receptor (CAR) T cell therapy represents a cutting-edge cancer treatment, making the development and testing of CAR T cells crucial for advancing this therapeutic strategy. We present a protocol for creating and characterizing human epidermal growth factor receptor 2 (HER2)- and glypican-3 (GPC3)-metabolic reprogramming (MR)-CAR T cells by overexpressing adenosine deaminase 1 (ADA1) and CD26 (also known as dipeptidylpeptidase-4 or DPP4). This approach effectively converts immunosuppressive adenosine into inosine, which supports T cell survival in glucose-deficient tumor microenvironments. The protocol includes producing retroviral vectors, generating CAR T cells, and conducting ecto-ADA1 activity, cytotoxicity, cell migration, and RNA sequencing assays. For complete details on the use and execution of this protocol, please refer to Hu et al.1.

Protocol for rapid and cost-effective extraction of genomic DNA from a wide range of wild yeast species for use in PCR-based applications.

Isolation of amplifiable genomic DNA is a prerequisite for the implementation of PCR-based techniques. Here we present a protocol for isolating the genomic DNA from a variety of wild yeast species. This can be completed in approximately 1 h and does not require sophisticated laboratory equipment. We describe steps for growing yeast cells, genomic data extraction, and downstream assay for amplification of specific sequences from the genomic DNA. We then detail procedures for gel electrophoresis and analysis of the results. For complete details on the use and execution of this protocol, please refer to Kristjuhan et al.1.

Protocol for preparation of a textile magnetoelastic generator patch.

The magnetoelastic generator (MEG) is a fundamentally new platform technology to convert mechanical motions into electrical signals for sensing, therapeutics, and energy applications. Here, we present a protocol for fabricating and characterizing the MEG for personalized muscle physiotherapy when integrated into a wearable textile patch. We describe the steps for fabricating such a textile MEG, including the magnetomechanical coupling (MC) and magnetic induction (MI) layers, and characterizing their magnetoelastic and electrical properties. We then detail procedures for monitoring muscle biomechanical activities and muscle physiotherapy application. For complete details on the use and execution of this protocol, please refer to Xu et al.1.

Protocol to generate a microfluidic vessels-on-chip platform using human pluripotent stem cell-derived endothelial cells.

Here, we present a protocol for producing a microfluidic vessel-on-chip platform using human pluripotent stem cell-derived endothelial cells (SC-ECs). We describe steps for manufacturing the 3D-printed chip, cell culturing to generate SC-ECs, hydrogel patterning, and the formation and cultivation of barrier-forming vessels. We then detail procedures for the retrieval of cells and media from the open microfluidic chip platform to enable multi-omics analysis. For complete details on the use and execution of this protocol, please refer to Marder et al.1.

A protocol for the detection of precision genome editing in human cells using One-pot DTECT.

Prime editing is a highly versatile CRISPR-based genome editing technology that allows for the precise installation of desired genetic variants. This protocol describes how to use One-pot DTECT to assess prime editing efficiency in human cells. Key steps include conducting prime editing, extracting genomic DNA, performing AcuI-tagging PCR, capturing genetic signatures, and detecting captured signatures through qualitative, quantitative, and visual methods. One-pot DTECT enables same-day detection of targeted genetic signatures introduced by precision genome editing technologies using off-the-shelf reagents. For complete details on the use and execution of this protocol, please refer to Baudrier et al.1.

Protocol for engineering binding domains to recognize ligand-bound receptors by using yeast surface display.

Yeast surface display is a versatile protein engineering technology, enabling precise control of the applied selection pressure. We present a yeast-surface-display-based protocol for the enrichment of binders specifically recognizing ligand-bound receptors. We describe steps for magnetic bead selections, random mutagenesis, and flow cytometric sorting, followed by library sequencing and detailed analysis of enriched clones. While this approach is exemplified with rcSso7d-based libraries and epidermal growth factor (EGF)-epidermal growth factor receptor (EGFR) complexes, it can also be adapted to other binder scaffolds and ligand-receptor systems. For complete details on the use and execution of this protocol, please refer to Dobersberger et al.1.

Protocol for monitoring phagocytosis of cancer cells by TAM-like macrophages using imaging cytometry.

Here, we present a protocol for monitoring phagocytosis by M2-type macrophages using automated counting of phagocytic events with an imaging cytometer. We describe steps for isolating and differentiating peripheral blood mononuclear cell (PBMC)-derived monocytes into M2-like macrophages, preparing cancer cells expressing a green fluorescence marker, labeling with a pH-sensitive dye, and co-culturing with macrophages. We then outline procedures for enumerating phagocytic events using an imaging cytometer. For complete details on the use and execution of this protocol, please refer to Mishra et al.1.

Protocol for screening and validating antibodies specific to protein phosphorylation sites using a set of yeast biopanning approaches.

Developing antibodies with high specificity against post-translationally modified epitopes remains a challenge. Yeast biopanning is well suited in screening for high-specificity binders. Here, we present a protocol for screening and validating antibodies specific to protein phosphorylation sites using a set of yeast biopanning approaches. We describe steps for screening a yeast surface display library for antibodies and other binders. We then detail procedures for validating the antibodies found by analyzing their specificity through whole-well image analysis in 96-well plates. For complete details on the use and execution of this protocol, please refer to Arbaciauskaite et al.1.

Protocol to create phenotypic primary human hepatocyte cultures using the RASTRUM 3D cell model platform.

To improve human hepatotoxicity prediction, in vitro liver cell models replicating hepatocyte function, drug metabolism, and toxicity are required. Here, we present a protocol for creating 3D primary human hepatocyte (PHH) cell models using the RASTRUM Platform. We describe the process for PHH model generation; procedures for characterizing the PHH model, including viability, albumin production, and CYP450 inducibility; and drug treatment using acetaminophen and troglitazone. This protocol has applications in upscaling phenotypic hepatotoxicity applications.

Protocol for in vivo nucleic acid delivery utilizing the rolling microneedle electrode array.

Electroporation temporarily enhances cell membrane permeability and promotes the absorption of external molecules. We have developed a device termed the rolling microneedle electrode array (RoMEA) that combines a densely arranged microneedle array of electrodes with rolling structures. Use RoMEA to create uniform skin micropores for efficient, low-damage transfection of nucleic acids over extended areas of the body. We describe in detail the design, fabrication, and assembly of the device and the application of in vivo electroporation of nucleic acids. For complete details on the use and execution of this protocol, please refer to Tongren Yang et al. 1.

Protocol for genetic engineering in Drosophila suzukii using microinjection.

The spotted wing Drosophila (Drosophila suzukii Matsumura) is recognized globally as a significant economic pest. Here, we present a protocol for genetic engineering in D. suzukii using microinjection. We describe steps for genetic engineering techniques, including transposon-mediated germline transformation, recombinase-mediated genome targeting, and CRISPR-mediated gene editing. This protocol can significantly expand the toolkit for functional genomics and genetic control studies of this pest. For complete details on the use and execution of this protocol, please refer to Schetelig and Handler,1 Schetelig et al.2 Yan et al.,3 and Yan et al.4.

Protocol to fabricate elastomer microwells for three-dimensional culture of primary adipocytes.

Our understanding of how adipocytes influence metabolic signaling, immune function, and cancer progression remains limited as the culture of primary adipocytes is challenging. Here, we present a protocol to fabricate elastomer microwells for three-dimensional culture of collagen-embedded adipocytes. We describe steps to cure and functionalize elastomer microwells and to isolate and embed primary adipocytes. We then detail how to culture and analyze adipocyte-collagen gels. This protocol provides broad applications to improve our understanding of adipocyte biology in health and disease.

Protocol for hybrid structured-light three-dimensional scanning and modeling of human postmortem brains.

Despite numerous neuropathological criteria for the evaluation of microscopic tissue slides, there are no systemic standards for sectioning postmortem human brain tissue. Here, we present a protocol for postmortem brain hybrid structured-light scanning to facilitate 3D printing of sectioning matrices. We describe steps for tissue preparation, 3D scanning and printing, and matrix-guided sectioning. This protocol can be used for streamlining tissue histopathology as well as in brain morphology volumetric analyses and applications in medical education. For complete details on the use and execution of this protocol, please refer to Barannikov et al.1.

Protocol for evaluating the effects of large gradient high magnetic fields on osteocyte function.

Osteocytes are the main mechanosensory cells and the primary regulators of bone metabolic homeostasis. Here, we present a protocol for evaluating the effects of the large gradient high magnetic field (LG-HMF) on osteocyte function. We describe steps for establishing a corresponding cell culture system in the LG-HMF generated by a superconducting magnet. We then detail procedures for using this cell culture system to study the effects of magnetic forces on the structure and function of murine long bone osteocyte Y4 cells. For complete details on the use and execution of this protocol, please refer to Zhang et al.1.

Protocol to probe how promoters decode TF dynamics in Saccharomyces cerevisiae by combining optogenetic control with microscopy.

Gene promoters filter transcription factor (TF) localization dynamics and changes in the DNA binding affinity of TFs. Here, we present a protocol to probe how promoters decode TF dynamics in Saccharomyces cerevisiae by combining optogenetic control with microscopy. We describe steps for preparing and characterizing a light delivery platform and light-controlled TF mutants. We then detail procedures for subjecting the TFs to light doses that generate defined patterns of localization while measuring fluorescent reporter gene activation via live-cell microscopy. For complete details on the use and execution of this protocol, please refer to Sweeney and McClean.1.

Protocol for isolating and identifying small extracellular vesicles derived from human umbilical cord mesenchymal stem cells.

Small extracellular vesicles (sEVs) are lipid bilayer-enclosed particles secreted by living cells. Here, we present a protocol for the collection and isolation of sEVs derived from human umbilical cord mesenchymal stem cells (hucMSCs). We describe steps for characterizing their morphology and integrity by transmission electron microscopy (TEM) and size distribution using nanoparticle tracking analysis (NTA) and an atomic force microscope (AFM). We then detail procedures for assessing nanoparticle size analysis and molecular markers by western blotting and Flow NanoAnalyzer.

Protocol for calculating binding free energy of RNA:RNA interactions through molecular dynamics simulations using adaptive biasing force technique.

The adaptive biasing force (ABF) technique allows sampling to proceed in a flat free energy surface when performing molecular dynamics (MD) simulations. Here, we present a protocol to perform MD simulations using the ABF technique and apply it to calculate the binding free energy of an RNA:RNA interaction. We describe steps for server setup, test running software, and building molecular models. We then detail procedures for running and configuring ABF-MD simulations and analyzing binding free energy and structural change. For complete details on the use and execution of this protocol, please refer to Fujita et al.1 and Kameda et al.2.

Protocol to use protein language models predicting and following experimental validation of function-enhancing variants of thymine-N-glycosylase.

Protein language models (PLMs) are machine learning tools trained to predict masked amino acids within protein sequences, offering opportunities to enhance protein function without prior knowledge of their specific roles. Here, we present a protocol for optimizing thymine-DNA-glycosylase (TDG) using PLMs. We describe steps for "zero-shot" enzyme optimization, construction of plasmids, double plasmid transfection, and high-throughput sequencing and data analysis. This protocol holds promise for streamlining the engineering of gene editing tools, delivering improved activity while minimizing the experimental workload. For complete details on the use and execution of this protocol, please refer to He et al.1.

Protocol for oleuropein-induced autophagy mediating drug tolerance in P. falciparum.

The anti-inflammatory activity of a phytocompound (oleuropein [OLP]) in the lipopolysaccharide (LPS)-mimicked macrophage model of inflammation demonstrates the importance of PI3K-Akt1 signaling in establishing "immune homeostasis." Here, we present a protocol for the cultivation of in vitro cultures of P. falciparum for carrying out drug sensitivity assays. We describe steps for parasite synchronization, drug treatment, DNA isolation, and starvation-induced autophagy. This protocol provides insights into autophagy and parasite tolerance to drug pressure. For complete details on the use and execution of this protocol, please refer to Sharma et al.1.

Protocol for profiling virus-to-host RNA-RNA interactions in infected cells by RIC-seq.

Virus-to-host RNA-RNA interactions directly regulate host mRNA stability and viral replication. However, globally profiling virus-to-host in situ RNA-RNA interactions remains challenging. Here, we present an RNA in situ conformation sequencing (RIC-seq)-based protocol for mapping high-confidence virus-to-host in situ RNA-RNA interactions in infected cells. We detail steps for formaldehyde crosslinking, pCp-biotin labeling, in situ proximity ligation, chimeric RNA enrichment, strand-specific library construction, and data analysis. This protocol allows unbiased identification of virus-to-host RNA-RNA interactions for various RNA viruses and is potentially applicable to DNA virus-derived transcripts. For complete details on the use and execution of this protocol, please refer to Zhao et al.1.