STAR Protocols最新文献

Regulating cancer-related microRNAs (miRNAs) may become a new generation of therapeutic modalities for cancer treatment. Here, we describe a protocol based on hepatoma-22 (H22) tumor-bearing mice to screen potential targets for treating hepatocellular carcinoma (HCC). We detail the construction of H22 tumor-bearing mice and treatment with two natural compounds, Ulva lactuca L. polysaccharide (ULP) and 5-fluorouracil (5FU). We further describe the isolation of the tumor tissues for miRNA sequencing and the discovery and validation of potential miRNA gene targets against HCC. For complete details on the use and execution of this protocol, please refer to Qiu et al.¹.

A combination of hematopoietic stem cell (HSC)- and endothelial cell (EC)-lineage-tracing mouse models enables us to determine blood cell origins. We present a protocol to induce cell labeling in vivo and to trace labeled hematopoietic cells to segregate their origins. We describe the steps for harvesting various hematopoietic tissues, antibody staining, and analyzing the Tomato+ percentages within each immune cell population. We also show how to estimate HSC- and EC-derived percentages of the target cell populations. For complete details on the use and execution of this protocol, please refer to Kobayashi et al.¹.

Here, we present a NAD⁺/NADH detection assay for evaluating NAD⁺, NADH, and NAD⁺/NADH ratio across diverse biological models, including Caenorhabditis elegans, mouse muscle tissue, mouse whole blood, and human whole blood. We describe steps for sample collection and preparation from different models as well as detection and calculation of NAD⁺ and NADH levels. This protocol is applicable for quantifying cellular/tissue NAD⁺ and NADH levels across different biological models.

We outline a protocol to visualize all mouse lower hindlimb skeletal muscles simultaneously. We describe procedures for orientating the whole lower hindlimb in gum tragacanth prior to freezing, simplifying the proceeding experimental steps, and enhancing the comprehensiveness of characterizations. We then detail steps for quantifying muscle fiber size and fiber type characteristics in a single cryosection using histochemistry and immunofluorescence. This protocol can be applied to histological and (immuno)histochemical evaluations such as muscle regeneration, fibrosis, enzymatic activity, and glycogen content.

Alveolar macrophages and other myeloid cells in the human airways are the primary cell types responding to respiratory pathogens. Here, we present a protocol for in vitro stimulation of cryopreserved human bronchoalveolar lavage (BAL) cells with mycobacterial antigens for phenotyping and quantifying proinflammatory cytokine responses in myeloid cells by mass cytometry. We demonstrate that the measure of markers of myeloid lineage and function is stable after freezing stained cells thereby allowing for batched analyses and/or machine downtime.

Patient-derived xenograft (PDX) models of acute myeloid leukemia (AML-PDX) offer advantages over cell line models by capturing the complexity and heterogeneity of patient-derived samples. Here, we present a protocol for developing a bioluminescent AML-PDX model in mice to evaluate chimeric antigen receptor (CAR) T cell therapy. We describe steps for transducing, engrafting, expanding, and enriching AML-PDX cells. We then detail procedures for in vitro and in vivo validation of the AML-PDX model for the evaluation of CAR T cell immunotherapy. For complete details on the use and execution of this protocol, please refer to Caulier et al.¹.

Calcium (Ca²⁺) imaging is a viable approach for imaging neuronal activity patterns in local brain circuits in living animals. Here, we present a protocol for gradient lens implantation in deep brain structures followed by in vivo Ca²⁺ imaging. We describe in detail the steps for surgery preparation, followed by lens implantation, setup for awake head-fixed imaging, and the recording process. For complete details on the use and execution of this protocol, please refer to Cherepanov et al.¹.

Lipophosphoglycan (LPG) is a macromolecule on the surface of Leishmania spp. parasites. The biochemical structure of LPG varies throughout the parasites' life cycle between proliferative (procyclic) and infective (metacyclic) stages, as well as between species and strains. Here, we outline a protocol for growing Leishmania parasites in vitro to harvest LPG. We describe steps for parasite differentiation and LPG extraction and purification. LPG has applications in medical research, such as in trained immunity and immunotherapy for cancer. For complete details on the use and execution of this protocol, please refer to dos Santos et al.¹.

Here, we present a protocol for quantifying microglial cell morphology at the single-cell level in mice. We provide comprehensive details, starting from optimal mouse brain dissection to computational analyses of up to 350 microglial cells per brain slice. Analyzing the morphology of microglial cells is essential for understanding their functional and activation states in different conditions, including during disease development and progression, as well as for assessing the effect of therapeutic interventions. For complete details on the use and execution of this protocol, please refer to Lind-Holm Mogensen et al.¹ and Fixemer et al.².

2-deoxy-D-glucose (2DG) is a glucose analog converted to 2-deoxy-D-glucose-6-phosphate (2DG-6P) by hexokinase in glycolysis. While 2DG commonly measures glucose uptake, 2DG-6P detects glucose utilization. Here, we present a protocol to measure glucose utilization in various tissues after entering a mouse's body using radiolabeled 2DG. We describe steps for preparing mice and chemicals, extracting blood, adding chemicals, and dissolving tissue. We then detail procedures for calculating glucose utilization using the trapezoid rule.