Flow cytometry is a valuable method for analyzing protein expressions at the single cell level but can be difficult to apply to large numbers of samples. This protocol provides instructions to perform a high-throughput small molecule screen using flow cytometry analysis of THP-1 cells, a human monocytic leukemia cell line. We describe a methodology for identifying compounds that regulate PD-L1 surface expression in IFN-γ-stimulated cells, which has been successfully used to screen a collection of ∼200,000 compounds. For complete details on the use and execution of this protocol, please refer to Zavareh et al. (2020).Two-dimensional (2D) culture of tumor cells fails to recapitulate some important aspects of cellular organization seen in in vivo experiments. In addition, cell cultures traditionally use non-physiological concentration of nutrients. Here, we describe a protocol for a facile three-dimensional (3D) culture format for cancer cells. This 3D platform helps overcome the 2D culture limitations. In addition, it allows for longitudinal modeling of responses to cancer therapeutics. For complete details on the use and execution of this protocol, please refer to Lhuissier et al. (2017), Lehmann et al. (2016), Liu et al. (2016), and Duval et al. (2011).The analysis of B cell receptors (BCR) from single B cells is crucial to understanding humoral immune responses. Here, we describe a protocol for the sequencing, cloning, and characterization of antibody genes that encode BCRs. We used this method to analyze the BCRs of different mouse B cell populations for somatic hypermutations, clonal and phylogenic relationships, and their affinity for cognate antigen. https://www.selleckchem.com/products/cwi1-2-hydrochloride.html For complete details on the use and execution of this protocol, please refer to Viant et al. (2020).Endothelial tip cells (ETCs) located at growing blood vessels display high morphological dynamics and associated intracellular Ca2+ activities with different spatiotemporal patterns during migration. Examining the Ca2+ activity and morphological dynamics of ETCs will provide an insight for understanding the mechanism of vascular development in organs, including the brain. Here, we describe a method for simultaneous monitoring and relevant analysis of the Ca2+ activity and morphology of growing brain ETCs in larval zebrafish. For complete details on the use and execution of this protocol, please refer to Liu et al. (2020).Controversies remain over the standard procedures for the modeling of skin fibrosis and its use in in vitro testing of different drugs. Here, we report a reproducible protocol for producing a skin fibrosis model using human dermal fibroblasts seeded in collagen hydrogel. Detailed procedures for the fabrication of cell/hydrogel constructs, fibrosis induction, protein extraction for western blotting analysis are presented along with how this model can be employed for investigating the possible anti-fibrotic functions of certain chemical compounds.Addressing bioenergetics is key to evaluate the impact of metabolism on the regulation of biological processes and its alteration in disease. Organoids are in vitro grown self-organizing structures derived from healthy and diseased tissue that recapitulate with high fidelity the tissue of origin. Bioenergetics is commonly analyzed by Seahorse XF analysis. However, its application to organoid studies is technically challenging. Here, we share our in-house optimized protocols to examine organoid bioenergetics in response to drugs, gene knockdown, or to characterize the metabolism of specific cell types. For complete details on the use and execution of this protocol, please refer to Ludikhuize et al. (2020).Major histocompatibility complexes (MHC) play a critical role in immunity by presenting peptides on the cell surface for T cell recognition. Identification of these peptides can be valuable to develop vaccines or immunotherapeutic strategies for infectious diseases and cancers. Mass spectrometry is the only tool available for unbiased identification of the immunopeptidome. Here, we describe a protocol for purification and identification of MHC class I peptides, including in-house purification of anti-MHC-antibody from hybridoma cells and the LC-MS/MS analysis of MHC-I bound peptides.Visualizing transcription live in Drosophila is providing important new insights into the spatiotemporal regulation of transcription. Here, we describe a protocol to visualize and quantitate transcription from gene loci that are tagged with MS2 stem-loop sequences in the Drosophila embryo. MS2 stem-loop sequences are recognized by a coat protein fused to a fluorescent protein and visualized with microscopy. We also describe an analysis pipeline to extract and subsequently quantify transcription dynamics. For complete details on the use and execution of this protocol, please refer to Hoppe et al. (2020).Micronuclei are aberrant nuclear compartments that form when chromosomes or chromosome fragments fail to incorporate into a primary nucleus during mitotic exit. Ruptures at the micronuclear envelope are associated with DNA damage and activation of immune sensing pathways. To gain insights into these processes, we have developed a method to purify ruptured micronuclei. This method paves the way toward understanding the consequences of micronuclear envelope rupture. For complete details on the use and execution of this protocol, please refer to Mohr et al. (2021).Double-stranded RNAs (dsRNAs) are abundantly present in cells, playing multiple regulatory functions. dsRNAs of viral origin activate innate immune responses. Since RNA editing and modifications affect the structure and recognition of RNAs, their alteration can result in the accumulation of aberrant endogenous dsRNAs inducing a deleterious innate immune response. Here, we present a complete protocol for the measurement of dsRNAs in a live mouse tissue using dsRNA immunoprecipitation and sequencing (dsRIP-Seq). This protocol focuses on tissue isolation, dsRNA immunoprecipitation and downstream computational analysis. For complete details on the use and execution of this protocol, please refer to Gao et al. (2020).