Notes

Non-Solvating and also Low-Dielectricity Cosolvent for Anion-Derived Strong Electrolyte Interphases inside Lithium Steel Power packs.
Bone marrow (BM) mesenchymal stromal cells play an important role in regulating stem cell quiescence and homeostasis; they are also key contributors to various hematological malignancies. However, human bone marrow stromal cells are difficult to isolate and prone to damage during isolation. This protocol describes a combination of mechanical and enzymatic isolation of BM stromal cells from human BM biopsies, followed by FACS sorting to separate stromal sub-populations including mesenchymal stromal cells, fibroblasts, and Schwann cells for single-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Leimkühler et al. (2020).Gnotobiology has revolutionized the study of microbiota-host interactions. This protocol explains how to generate, maintain, and monitor gnotobiotic mice. Three monitoring methods are presented and compared bacterial culture, microscopy to visualize the presence (or absence) of bacteria using Gram staining or DNA staining, and 16S rRNA gene amplification and sequencing. The generation and maintenance of gnotobiotic animals should be performed in a germ-free and gnotobiotic facility to guarantee sterility and precision of gnotobiotic conditions. For complete details on the use and execution of this protocol, please refer to McDonald et al., 2020.Reactive oxygen species (ROS) are important signaling molecules for physiologic processes such as acute response to hypoxia. However, reliable real-time ROS measurement in cells has been a long-standing methodological challenge. Here, we present a protocol to record acute changes in ROS production in sensory cells from mouse carotid body (CB) slices using redox-sensitive green fluorescent protein probes and microfluorimetry. This protocol provides sensitive and reproducible quantification of ROS during acute hypoxia in different subcellular compartments of CB glomus cells. For complete details on the use and execution of this protocol, please refer to Fernández-Agüera et al. Blebbistatin (2015) and Arias-Mayenco et al. (2018).Tumor organoids and patient-derived orthotopic xenografts (PDOXs) are some of the most valuable pre-clinical tools in cancer research. In this protocol, we describe efficient derivation of organoids and PDOX models from glioma patient tumors. We provide detailed steps for organoid culture, intracranial implantation, and detection of tumors in the brain. We further present technical adjustments for standardized functional assays and drug testing. For complete details on the use and execution of this protocol, please refer to Golebiewska et al. (2020).Mitochondrial metabolism is a critical mechanism that is deregulated in numerous retinal diseases. Here, we elaborate a protocol to quantify oxygen consumption rate as a measure of mitochondrial respiration directly from mouse retinal tissue pieces. Our procedure combines the use of Seahorse extracellular flux technology and ex vivo retinal tissue isolation and is robustly reproducible under different treatment conditions. This protocol allows direct assessment of mitochondrial function in response to drug treatments or genetic manipulation in mouse models. For complete details on the use and execution of this protocol, please refer to Shetty et al. (2020), Sardar Pasha et al. (2021), Kooragayala et al. (2015), and Joyal et al. (2016).This protocol describes the use of the deterministic barcoding in tissue for spatial omics sequencing platform to construct a multi-omics atlas on fixed frozen tissue samples. This approach uses a microfluidic-based method to introduce combinatorial DNA oligo barcodes directly to the cells in a tissue section fixed on a glass slide. This technique does not directly resolve single cells but can achieve a near-single-cell resolution for spatial transcriptomics and spatial analysis of a targeted panel of proteins. For complete details on the use and execution of this protocol, please refer to Liu et al. (2020).Preparation of long single-stranded (ss)DNA in large quantities with high efficiency and purity remains a synthetic challenge. Here, we present a protocol for using DNA-hydrolyzing DNA enzymes (deoxyribozymes) for efficient biotechnological production of milligrams of ssDNA with a customizable sequence up to a few kilobases. Our protocol provides a convenient yet economical way to store the sequence information of target ssDNA on phages for selective mass production on demand. For complete details on the use and execution of this protocol, please refer to Jia et al. (2021).Correlation of 3D images acquired on different microscopes can be a daunting prospect even for experienced users. This protocol describes steps for registration of images from soft X-ray absorption contrast imaging and super-resolution fluorescence imaging of hydrated biological materials at cryogenic temperatures. Although it is developed for data generated at synchrotron beamlines that offer the above combination of microscopies, it is applicable to all analogous imaging systems where the same area of a sample is examined using successive non-destructive imaging techniques. For complete details on the use and execution of this protocol, please refer to Kounatidis et al. (2020).Here, we present a revised protocol to derive neuroepithelial stem (NES) cells from human induced pluripotent stem cells. NES cells can be further differentiated into a culture of neurons (90%) and glia (10%). We describe how to derive and maintain NES cells in culture and how to differentiate them. In addition, we show the potential use of NES cells to study the role of reactive oxygen species in neuronal differentiation and a guideline for NES cell transfection. For complete details on the use and execution of this protocol, please refer to Calvo-Garrido et al. (2019); Falk et al. (2012).Small molecular probes designed for photopharmacology and opto-chemogenetics are rapidly gaining widespread recognition for investigations of transient receptor potential canonical (TRPC) channels. This protocol describes the use of three photoswitchable diacylglycerol analogs-PhoDAG-1, PhoDAG-3, and OptoDArG-for ultrarapid activation and deactivation of native TRPC2 channels in mouse vomeronasal sensory neurons and olfactory type B cells, as well as heterologously expressed human TRPC6 channels. Photoconversion can be achieved in mammalian tissue slices and enables all-optical stimulation and shutoff of TRPC channels. For complete details on the use and execution of this protocol, please refer to Leinders-Zufall et al. (2018).
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