TY - JOUR AB - The pig is a valuable animal model in diabetes research; however, standardized protocols are essential for evaluating in vivo metabolism. Here, we present a protocol for in vivo assessment of glucose control and insulin secretion and sensitivity in the pig. We describe steps for catheter implantation, testing of intravenous glucose tolerance, performance of hyperinsulinemic-euglycemic clamps (HECs) and hyperglycemic clamps (HGCs), and blood processing. We then detail procedures for analysis of plasma glucose, insulin, glucagon, and C-peptide concentrations as well as data analysis. For complete details on the use and execution of this protocol, please refer to Renner et al.1 and Renner et al.2. AU - Eckstein, Y.* AU - Kessler, B.* AU - Hinrichs, A.* AU - Novak, I.* AU - von Thaden, A.* AU - Lorenzen, T.* AU - Rathkolb, B. AU - Scholz, A.* AU - Blutke, A.* AU - Koopmans, S.J.* AU - Hrabě de Angelis, M. AU - Christoffersen, B.* AU - Wolf, E.* AU - Renner, S.* C1 - 74143 C2 - 57086 CY - Radarweg 29, 1043 Nx Amsterdam, Netherlands TI - Protocol for in vivo assessment of glucose control and insulin secretion and sensitivity in the pig. JO - STAR Protoc. VL - 6 IS - 2 PB - Elsevier PY - 2025 ER - TY - JOUR AB - Here, we present a protocol to generate key pancreatic cell types in vitro using human pluripotent stem cell (hPSC)-based Matrigel-overlay organoid differentiation. These include multipotent and bipotent progenitors, endocrine progenitors, and hormone-producing endocrine cells. We describe steps for culturing hPSCs as a 2D monolayer, applying a Matrigel overlay to create a 3D epithelial niche, and guiding stepwise differentiation. This system supports live-cell imaging and real-time tracking of morphogenesis and fate decisions, providing a platform for studying organ development and disease. For complete details on the use and execution of this protocol, please refer to Ulf et al.1. AU - Tian, C. AU - Tiemann, U.* AU - Hermann, F.* AU - Semb, H. C1 - 74778 C2 - 57574 CY - Radarweg 29, 1043 Nx Amsterdam, Netherlands TI - Protocol for in vitro modeling of specification and morphogenesis of early pancreas development using human pluripotent stem cell-based organoid differentiation. JO - STAR Protoc. VL - 6 IS - 2 PB - Elsevier PY - 2025 ER - TY - JOUR AB - To unravel the complexity of biological processes, it is necessary to resolve the underlying protein organization down to single proteins. Here, we present a protocol for secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a DNA-PAINT-based super-resolution microscopy technique that is capable of resolving virtually unlimited protein species with single-protein resolution. We describe the steps to prepare neuronal cultures, troubleshoot and conduct SUM-PAINT experiments, and analyze the resulting feature-rich neuronal cell atlases using unsupervised machine learning approaches. For complete details on the use and execution of this protocol, please refer to Unterauer et al.1. AU - Unterauer, E.M.* AU - Schentarra, E.M.* AU - Jevdokimenko, K.* AU - Shetab Boushehri, S. AU - Marr, C. AU - Opazo, F.* AU - Fornasiero, E.F.* AU - Jungmann, R.* C1 - 73591 C2 - 57122 CY - Radarweg 29, 1043 Nx Amsterdam, Netherlands TI - Protocol for SUM-PAINT spatial proteomic imaging generating neuronal architecture maps in rat hippocampal neurons. JO - STAR Protoc. VL - 6 IS - 1 PB - Elsevier PY - 2025 ER - TY - JOUR AB - Chromatin immunoprecipitation (ChIP) combined with sequencing has revolutionized our understanding of gene regulation; however, its application to frozen adipose tissue presents unique challenges due to the high levels of lipid content. Here, we present a protocol for ChIP of histone modifications in human frozen adipose tissue. We describe steps for tissue preparation, chromatin isolation, sonication, pre-clearing of chromatin, and immunoprecipitation. We then detail procedures for elution, crosslink reversal, chromatin purification, quality control, and library synthesis. AU - Cayir, A.* AU - Tannæs, T.M.* AU - Saeed, S.* AU - Blüher, M. AU - Böttcher, Y.* C1 - 70942 C2 - 55980 CY - Radarweg 29, 1043 Nx Amsterdam, Netherlands TI - Protocol for chromatin immunoprecipitation of histone modifications in frozen adipose tissue. JO - STAR Protoc. VL - 5 IS - 3 PB - Elsevier PY - 2024 ER - TY - JOUR AB - 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. AU - Remmert, C. AU - Otgonbayar, M. AU - Perschel, J.A. AU - Marder, M. AU - Meier, M. C1 - 71650 C2 - 56333 CY - Radarweg 29, 1043 Nx Amsterdam, Netherlands TI - Protocol to generate a microfluidic vessels-on-chip platform using human pluripotent stem cell-derived endothelial cells. JO - STAR Protoc. VL - 5 IS - 3 PB - Elsevier PY - 2024 ER - TY - JOUR AB - A major impediment to effective cellular therapies in solid tumors is the limited access of therapeutic cells to the tumor site. One strategy to overcome this challenge is to endow T cells with chemotactic properties required to access tumor tissue. Here, we present a chimeric antigen receptor (CAR)-modified T cell strategy centered around enhanced T cell trafficking. We outline isolation, activation, and transduction of human T cells, as well as techniques for assessing migratory and cytotoxic capacity of CAR-T cells. For complete details on the use and execution of this protocol, please refer to Lesch et al. (2021). AU - Oner, A.* AU - Kobold, S. C1 - 66259 C2 - 53122 CY - Radarweg 29, 1043 Nx Amsterdam, Netherlands TI - Transwell migration assay to interrogate human CAR-T cell chemotaxis. JO - STAR Protoc. VL - 3 IS - 4 PB - Elsevier PY - 2022 ER - TY - JOUR AB - Human endogenous retroviruses (HERVs) comprise many regulatory elements and can regulate host gene activity at different expression levels via multiple mechanisms. Here, we introduce a step-by-step protocol to activate or repress transcription of HERV-K(HML-2) elements using the CRISPRa and CRISPRi technologies in human embryonic stem cells. This protocol can help deciphering the functional role of HERV-K(HML-2) elements in critical biological processes. The protocol may easily be adapted to other cell lines and HERV groups with relatively low sequence heterogeneity. For complete details on the use and execution of this protocol, please refer to Padmanabhan Nair et al. (2021). AU - Padmanabhan Nair, V. AU - Mayer, J.* AU - Vincendeau, M. C1 - 64778 C2 - 52452 TI - A protocol for CRISPR-mediated activation and repression of human endogenous retroviruses in human pluripotent stem cells. JO - STAR Protoc. VL - 3 IS - 2 PY - 2022 ER - TY - JOUR AB - We present a protocol to characterize the morphological properties of individual neurons reconstructed from microscopic imaging. We first describe a simple procedure to extract relevant morphological features from digital tracings of neural arbors. Then, we provide detailed steps on classification, clustering, and statistical analysis of the traced cells based on morphological features. We illustrate the pipeline design using specific examples from zebrafish anatomy. Our approach can be readily applied and generalized to the characterization of axonal, dendritic, or glial geometry. For complete context and scientific motivation for the studies and datasets used here, refer to Valera et al. (2021). AU - Bijari, K.* AU - Valera, G. AU - López-Schier, H. AU - Ascoli, G.A.* C1 - 63272 C2 - 51447 TI - Quantitative neuronal morphometry by supervised and unsupervised learning. JO - STAR Protoc. VL - 2 IS - 4 PY - 2021 ER - TY - JOUR AB - The recapitulation of human developmental processes and pathological manifestations requires access to specific cell types and precursor stages during embryogenesis and disease. Here, we describe a scalable in vitro differentiation protocol to guide human pluripotent stem cells stepwise into pancreatic duct-like organoids. The protocol mimics pancreatic duct development and was successfully used to model the onset and progression of pancreatic ductal adenocarcinoma; the approach is suitable for multiple downstream applications. However, the protocol is cost- and time-intensive. For complete details on the use and execution of this protocol, please refer to Breunig et al. (2021). AU - Breunig, M.* AU - Merkle, J.* AU - Melzer, M.K.* AU - Heller, S.* AU - Seufferlein, T.* AU - Meier, M. AU - Hohwieler, M.* AU - Kleger, A.* C1 - 63769 C2 - 51625 TI - Differentiation of human pluripotent stem cells into pancreatic duct-like organoids. JO - STAR Protoc. VL - 2 IS - 4 PY - 2021 ER - TY - JOUR AB - Quantifying differential genome occupancy by chromatin immunoprecipitation (ChIP) remains challenging due to variation in chromatin fragmentation, immunoprecipitation efficiencies, and intertube variability. In this protocol, we add heterologous spike-ins from Drosophila chromatin as an internal control to the mice chromatin before immunoprecipitation to normalize for technical variation in ChIP-qPCR or ChIP-seq. The choice of spike-in depends on the evolutionary conservation of the protein of interest and the antibody used. For complete details on the use and execution of this protocol, please refer to Greulich et al. (2021). AU - Greulich, F.* AU - Mechtidou, A. AU - Horn, T. AU - Uhlenhaut, N.H. C1 - 62304 C2 - 50602 TI - Protocol for using heterologous spike-ins to normalize for technical variation in chromatin immunoprecipitation. JO - STAR Protoc. VL - 2 IS - 3 PY - 2021 ER - TY - JOUR AB - We have developed a protocol to quantify the position of a cell in a branched structure based on two-dimensional microscopy images of tissue sections. Biological branched structures include organs such as the lungs, kidneys, and pancreas. In these organs, cell fate has been correlated with position, based on a qualitative estimate. However, a quantitative means of evaluating the cell position has been lacking. With this protocol, the correlation between cell fate and cell position was measured in mouse embryonic pancreas. For complete details on the use and execution of this protocol, please refer to Nyeng et al. (2019). AU - Heilmann, S.* AU - Semb, H. AU - Nyeng, P.* C1 - 63287 C2 - 51454 TI - Quantifying spatial position in a branched structure in immunostained mouse tissue sections. JO - STAR Protoc. VL - 2 IS - 4 PY - 2021 ER - TY - JOUR AB - Isolation of nuclei tagged in specific cell types (INTACT) allows for stress-free and high-throughput analyses of cellular subpopulations. Here, we present an improved protocol for isolation of pure and high-quality GFP-labeled nuclei from frozen livers of INTACT mice, as well as protocols for downstream sequencing analyses. The adaptation to frozen tissue provides a pause point that allows sampling at multiple time points and/or phenotypic characterization of livers prior to nuclei isolation and downstream analyses. For complete details on the use of this protocol, please refer to Loft et al. (2021). AU - Herzig, S. AU - Schmidt, S.F. AU - Loft, A. C1 - 63050 C2 - 51244 TI - Purification of GFP-tagged nuclei from frozen livers of INTACT mice for RNA- and ATAC-sequencing. JO - STAR Protoc. VL - 2 IS - 3 PY - 2021 ER - TY - JOUR AB - With the advancement of laser-based microscopy tools, it is now possible to explore mechano-kinetic processes occurring inside the cell. Here, we describe the advanced protocol for studying the DNA repair kinetics in real time using the laser to induce the DNA damage. This protocol can be used for inducing, testing, and studying the repair mechanisms associated with DNA double-strand breaks, interstrand cross-link repair, and single-strand break repair. For complete details on the use and execution of this protocol, please refer to Kumar et al. (2017, 2020). AU - Madhavan, B.K.* AU - Han, Z.* AU - Sickmann, A.* AU - Pepperkok, R.* AU - Nawroth, P.P. AU - Kumar, V.* C1 - 62898 C2 - 51152 TI - A laser-mediated photo-manipulative toolbox for generation and real-time monitoring of DNA lesions. JO - STAR Protoc. VL - 2 IS - 3 PY - 2021 ER - TY - JOUR AB - We here report a flow-cytometry-based protocol to measure single-cell protein expression in small samples. The protocol is optimized for simultaneous detection of fluorescent proteins and intracellular and surface antigens in the embryonic pancreas from the mouse. Owing to low cell numbers, current protocols for flow cytometric analysis of embryonic tissues rely on tissue pooling. Our protocol enables analysis of one pancreas per sample, thereby facilitating detection of biological variation and minimizing the number of experimental animals needed. For complete details on the use and execution of this protocol, please refer to Nyeng et al (2019). AU - Nyeng, P.* AU - Dela Cruz, G.V.* AU - Semb, H. C1 - 62579 C2 - 50955 TI - Flow cytometry detection of surface and intracellular antigens in pancreas from a single mouse embryo. JO - STAR Protoc. VL - 2 IS - 3 PY - 2021 ER - TY - JOUR AB - This protocol describes an easy and reliable in-gel proteasome assay to quantify the activity and composition of different proteasome complexes in cells and tissues. The assay works well with limited amounts of total cell protein lysates. Although this assay is optimized specifically for the proteasome chymotrypsin-like activity, it can be expanded to other proteasome activities as well. Using antibodies that detect distinct proteasome subunits or regulators, we can determine the composition and relative quantity of active proteasome complexes. For complete details on the use and execution of this protocol, please refer to Meul et al. (2020). AU - Yazgili, A.S. AU - Meul, T. AU - Welk, V. AU - Semren, N. AU - Kammerl, I.E. AU - Meiners, S. C1 - 61999 C2 - 50562 TI - In-gel proteasome assay to determine the activity, amount, and composition of proteasome complexes from mammalian cells or tissues. JO - STAR Protoc. VL - 2 IS - 2 PY - 2021 ER - TY - JOUR AB - The budding yeast Saccharomyces cerevisiae is an excellent model organism to dissect the maintenance and inheritance of phenotypes due to its asymmetric division. This requires following individual cells over time as they go through divisions to define pedigrees. Here, we provide a detailed protocol for collecting and analyzing time-lapse imaging data of yeast cells. The microfluidics protocol can achieve improved time resolution for single-cell tracking to enable characterization of maintenance and inheritance of phenotypes. For complete details on the use and execution of this protocol, please refer to Bheda et al. (2020a). AU - Bheda, P. AU - Aguilar-Gómez, D.* AU - Kukhtevich, I. AU - Becker, J.* AU - Charvin, G.* AU - Kirmizis, A.* AU - Schneider, R. C1 - 60959 C2 - 49755 TI - Microfluidics for single-cell lineage tracking over time to characterize transmission of phenotypes in Saccharomyces cerevisiae. JO - STAR Protoc. VL - 1 IS - 3 PY - 2020 ER -