Protocol for isolating leukemia-derived extracellular vesicles from the spleen of preclinical models of leukemia using ultracentrifugation.

Here, we present a protocol for the direct isolation of small extracellular vesicles (sEVs) from the spleen of preclinical murine models of leukemia using ultracentrifugation. We describe steps for tissue collection, sample preparation, ultracentrifugation-based isolation, and sEV characterization. This protocol allows for efficient enrichment of both leukemia and its microenvironment-derived sEV (LME-sEV), providing a valuable tool for studying their composition and functional roles. Potential applications include investigating the role of sEV in leukemia progression and identifying biomarkers. For complete details on the use and execution of this protocol, please refer to Gargiulo et al.1.

Protocol for the isolation and purification of endoplasmic reticulum-plasma membrane junctions from the mouse brain.

Dynamic communication between intracellular organelles often takes place at specialized membrane contact sites that form between their membranes. Here we detail a procedure for the purification of endoplasmic reticulum-plasma membrane (ER-PM) junctions from the mouse brain. We describe steps for homogenizing isolated brain hemispheres and sequential centrifugation to remove the nuclear fraction from the other membrane fractions. We then detail procedures for separating the resulting crude membrane fractions by sucrose density gradients and purifying into their respective pellets. For complete details on the use and execution of this protocol, please refer to Weesner et al.1.

Protocol for isolating small cytosolic dsDNA from cultured murine cells.

We recently identified a class of small cytosolic double-stranded DNA (scDNA) approximately 20-40 bp in size in human and mouse cells. Here, we present a protocol for scDNA isolation from cultured murine cells. We describe steps for cytosolic compartment separation, DNA isolation in the cytosolic fraction using phenol-chloroform extraction, and ethanol precipitation. We then detail procedures for denaturing purified cytosolic DNA through urea polyacrylamide gel electrophoresis and obtaining scDNA in the cytosolic DNA fraction via gel purification. For complete details on the use and execution of this protocol, please refer to Liu et al.1.

Protocol to identify S-acylated proteins in hippocampal neurons using ω-alkynyl fatty acid analogs and click chemistry.

S-acylation, commonly palmitoylation, is the addition of fatty acids to cysteines to regulate protein localization and function. S-acylation detection has been hampered by limited sensitivity and selectivity in low-protein, costly samples like cultured neurons. Here, we present a protocol for sensitive and selective bioorthogonal labeling and click-chemistry-based detection of S-acylated proteins in primary hippocampal neurons. We describe steps for metabolically labeling neurons with alkynyl fatty acid, click chemistry, NeutrAvidin-based capture, and elution with hydroxylamine.

Protocol for sorting and culturing of mouse early erythroid progenitor BFU-E cells.

Techniques allowing the long-term culture of the burst-forming unit of erythroid (BFU-E) progenitor cells are essential for understanding erythropoiesis. Here, we present a protocol for sorting mouse BFU-E cells and culturing them in a medium that promotes BFU-E cell expansion. We describe steps for isolating BFU-E cells from mouse fetal livers by combining magnetic microbeads with flow cytometry and culturing BFU-E cells with a specific expansion media. This approach can enhance the production of BFU-E cells. For complete details on the use and execution of this protocol, please refer to Li et al..1.

An optimized protocol for the extraction and quantification of cytosolic DNA in mammalian cells.

Leakage of mitochondrial or nuclear DNA into the cytosol can occur following viral infections, radiation damage, and some cancers. Here, we present an optimized protocol for isolating and quantifying cytosolic DNA from mammalian cells. We describe steps for collecting cytosolic fractions from cells, extracting DNA using columns, and quantifying extracted DNA using qPCR. This straightforward protocol can be completed in as little as 5 hours, and allows for the identification of the source of DNA. For complete details on the use and execution of this protocol, please refer to Jahun et al.1.

Protocol for immunodot blot detection of UVB photoproducts in extracellular DNA.

UV radiation induces the formation of adducts in genomic DNA within cells that are later found to be present in cell-free fractions associated with extracellular vesicles (EVs) outside of cells. Here, we present a protocol for isolating UV photoproducts in extracellular DNA released from UVB-irradiated cells via differential centrifugation. We then detail steps for monitoring the DNA adducts using DNA immunoblotting. This protocol can be applied for detection of DNA adducts in EVs from cell culture and skin explant models. For complete details on the use and execution of this protocol, please refer to Carpenter et al.1.

Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing.

Adipocyte size and fragility and commercial kit costs impose significant limitations on single-cell RNA sequencing of adipose tissue. Accordingly, we developed a workflow to isolate and sample-barcode nuclei from individual adipose tissue samples, integrating flow cytometry for quality control, counting, and precise nuclei pooling for direct loading onto the popular 10× Chromium controller. This approach can eliminate batch confounding, and significantly reduces poor-quality nuclei, ambient RNA contamination, and droplet loading-associated reagent waste, resulting in pronounced improvements in information content and cost efficiency.

A protocol for measuring the activity of protein kinase C-delta in murine bone-marrow-derived dendritic cells.

Protein kinase C-δ (PKC-δ) is a key enzyme controlling growth, differentiation, and apoptosis in various cells, including immune cells. Here, we present a protocol to determine PKC-δ activation in response to increased membrane-bound diacylglycerol or phorbol-12-myristate-13-acetate treatment in murine bone-marrow-derived dendritic cells. We describe steps for dendritic cell differentiation, the isolation of plasma membrane lipids, and the quantification of diacylglycerol. We then detail procedures for measuring PKC-δ kinase activity by in vitro assay, indirect immunofluorescence, and western blotting experiments. For complete details on the use and execution of this protocol, please refer to Parsons et al.1.

Protocol for the derivation and alveolar type 2 differentiation of late-stage lung tip progenitors from the developing human lungs.

Molecular and cellular mechanisms of human lung alveolar development are poorly understood due to a lack of in vitro model systems. This protocol details the isolation, derivation, and genetic modification of lung tip epithelial progenitors from human fetal lungs. It includes steps for isolating distal lung epithelial cells, expanding tip progenitor organoids, culturing tip organoids in vitro, and differentiating them into alveolar type 2 cells. This will aid in understanding alveolar differentiation mechanisms and neonatal diseases. For complete details on the use and execution of this protocol, please refer to Lim et al.1.

Protocol for simultaneous isolation of high-quality and high-quantity cardiomyocytes and non-myocyte cells from adult rat hearts.

Isolating high-quality different cell types is a powerful approach for understanding cellular compositions and features in the heart, but it is challenging. The available protocols typically focus on isolating one or two cell types. Here, we present a protocol to simultaneously isolate high-quality and high-quantity cardiomyocytes and non-myocyte cells, including immune cells, from adult rat hearts. We describe steps for purifying cells using bovine serum albumin. We also detail procedures for viability analysis and cell type identification using fluorescence-activated cell sorting. For complete details on the use and execution of this protocol, please refer to Zhang et al.,1 Valkov et al.,2 Vang et al.,3 and Li et al.4.

Protocol for qualitative analysis of lysosome immunoprecipitation from patient-derived glioblastoma stem-like cells.

Lysosomes are critical for the sustenance of glioblastoma stem-like cells (GSCs) properties. We present a protocol to enrich and purify lysosomes from patient-derived GSCs in culture. We describe the steps required to stably express a tagged lysosomal protein in GSCs, mechanically lyse cells, magnetically immunopurify lysosomes, and qualitatively assess these organelles. We then detail the procedure for retrieving intact and purified lysosomes from GSCs. We also specify cell culture conditions, storage procedures, and sample preparation for immunoblotting. For complete details on the use and execution of this protocol, please refer to Maghe et al.1.

Protocol for the isolation and single-nuclei multiomic analyses of the human fetal epicardium.

The characterization of cell populations that reside in the outer layer of the heart has been hindered by difficulties in their isolation. Here, we present a protocol for isolation and single-nuclei multiomic analyses of the human fetal epicardium. We describe steps for microdissection, isolation, and enrichment of epicardial cells by mechanical dissociations and direct lysis. We then detail procedures for integrating transcriptome and chromatin accessibility datasets. This approach allows the analysis of diverse cell populations, marked by unique cis-regulatory elements. For complete details on the use and execution of this protocol, please refer to Travisano et al.1.

Protocol to identify DNA-binding proteins recognizing nucleotide repeat dsDNAs.

DNA-binding proteins perform diverse functions, including regulating cellular growth and orchestrating chromatin architecture. Here, we present a protocol to discover proteins specifically interacting with a hexanucleotide repeat DNA, the expansion of which is known as the most frequent genetic cause of familial C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia. We describe steps to fish out DNA-binding proteins recognizing double-stranded repeat DNAs using a SILAC (stable isotope labelling by amino acids in cell culture)-based approach and validate the results using electrophoretic mobility shift assay. For complete details on the use and execution of this protocol, please refer to Liu et al.1.

Isolating plasma extracellular vesicles from mouse blood using size-exclusion chromatography, density gradient, and ultracentrifugation.

Circulating extracellular vesicles (EVs) could serve for the surveillance of diverse pathological conditions. We present a protocol for enriching and isolating plasma EVs from mouse blood. We describe steps for employing ultracentrifugation, size-exclusion chromatography, and density gradients, required for further quantitative and qualitative analysis. We detail the procedure for retrieving optimal volume of blood while preserving its integrity and avoiding hemolysis. We also describe the preparation of EVs from this complex fluid containing soluble proteins, aggregates, and lipoprotein particles. For complete details on the use and execution of this protocol, please refer to André-Grégoire et al. (2022).1.

Protocol to isolate RBP-mRNA complexes using RNA-CLIP and examine target mRNAs.

RNA-binding proteins (RBPs) regulate diverse functions by interacting with target transcripts. Here we present a protocol to isolate RBP-mRNA complexes using RNA-CLIP and examine target mRNAs in association with ribosomal populations. We describe steps to identify specific RBPs and RNA targets reflecting a variety of developmental, physiological, and pathological states. This protocol enables RNP complex isolation from tissue sources (liver and small intestine) or populations of primary cells (hepatocytes), but not at a single-cell level. For complete details on the use and execution of this protocol, please refer to Blanc et al. (2014)1 and Blanc et al. (2021).2.

Optimized protocol for quantification of mitochondrial non-heme and heme iron content in mouse tissues and cultured cells.

Impaired mitochondrial iron metabolism is associated with aging and a variety of diseases, and there is a growing need to accurately quantify mitochondrial iron levels. This protocol provides an optimized method for evaluating non-heme and heme iron in mitochondrial and cytosolic fractions of tissues and cultured cells. Our protocol consists of three steps: sample fractionation, non-heme iron measurement, and heme iron measurement. For complete details on the use and execution of this protocol, please refer to Sato et al. (2022).1.

Fast and efficient synaptosome isolation and post-synaptic density enrichment from hiPSC-motor neurons by biochemical sub-cellular fractionation.

We describe here a time-efficient, in-house protocol for synaptosome isolation and enrichment of the post-synaptic density (PSD) from hiPSC-derived motor neurons. By using biochemical sub-cellular fractionation, the crude synaptosome is first isolated from the cytosol and is then further separated into the synaptic cytosol and the enriched PSD fraction. The protocol can also potentially be adapted to other hiPSC-derived neuronal types, with necessary changes made to cell seeding density and buffer volumes.

Protocol for mitochondrial isolation and sub-cellular localization assay for mitochondrial proteins.

Here, we provide a protocol to isolate mitochondria from cultured cells and extract differently located mitochondrial proteins. We detail steps to separate both integral and peripheral membrane proteins from soluble proteins using sonication. We describe the separation of integral membrane proteins from the peripheral membrane and soluble proteins using sodium carbonate extraction. Furthermore, we detail the use of proteinase K and Triton X-100 to distinguish outer membrane proteins from mitochondrial proteins.

Protocol for isolation of microbiota-derived membrane vesicles from mouse blood and colon.

Bacterial membrane vesicles have emerged as gadgets allowing remote communication between the microbiota and distal host organs. Here we describe a protocol for enriching vesicles from serum and colon that could widely be adapted for other tissues. We detail pre-clearing of serum or colon fluids using 0.2-µm syringe filters and their concentration by centrifugal filter devices. We also describe vesicle isolation with qEV size exclusion columns and finally the concentration of isolated vesicle fractions for downstream analyses. For complete details on the use and execution of this protocol, please refer to Erttmann et al. (2022).1.