Protocols in Current Issue
    Generating Three-dimensional Human Granulomas in vitro to Study Mycobacterium tuberculosis-host Interaction
    Authors:  Ainhoa Arbués, Michael Kammüller and Damien Portevin, date: 11/20/2020, view: 446, Q&A: 0
    [Abstract] Granulomas are organized multicellular structures that constitute the hallmark of an infection by the human pathogen Mycobacterium tuberculosis (Mtb). A better understanding of the complex host-Mtb interactions within the granuloma’s environment may lead to new therapeutic or preventive tools to improve the control of the ...
    Advances in Proximity Ligation in situ Hybridization (PLISH)
    Authors:  Monica Nagendran, Adam M Andruska, Pehr B Harbury and Tushar J Desai, date: 11/05/2020, view: 695, Q&A: 0
    [Abstract] Understanding tissues in the context of development, maintenance and disease requires determining the molecular profiles of individual cells within their native in vivo spatial context. We developed a Proximity Ligation in situ Hybridization technology (PLISH) that enables quantitative measurement of single cell gene expression ...
    In vitro Glutamylation Inhibition of Ubiquitin Modification and Phosphoribosyl-Ubiquitin Ligation Mediated by Legionella pneumophila Effectors
    Authors:  Alan G. Sulpizio, Marena E. Minelli and Yuxin Mao, date: 11/05/2020, view: 243, Q&A: 0

    Glutamylation is a posttranslational modification where the amino group of a free glutamate amino acid is conjugated to the carboxyl group of a glutamate side chain within a target protein. SidJ is a Legionella kinase-like protein that has recently been identified to perform protein polyglutamylation of the Legionella SdeA Phosphoribosyl-Ubiquitin

    Derivation of Induced Pluripotent Stem Cells from Human Fibroblasts Using a Non-integrative System in Feeder-free Conditions
    Authors:  Alvaro A. Beltran, Sarahi G. Molina and Adriana S. Beltran, date: 10/20/2020, view: 337, Q&A: 0
    [Abstract] Induced pluripotent stem cells (iPSCs) are genetically reprogrammed somatic cells that exhibit features identical to those of embryonic stem cells (ESCs). Multiple approaches are available to derive iPSCs, among which the Sendai virus is the most effective at reprogramming different cell types. Here we describe a rapid, efficient, safe, and ...
    Quantification of Salivary Charged Metabolites Using Capillary Electrophoresis Time-of-flight-mass Spectrometry
    Authors:  Masahiro Sugimoto, Sana Ota, Miku Kaneko, Ayame Enomoto and Tomoyoshi Soga, date: 10/20/2020, view: 419, Q&A: 0
    [Abstract] Salivary metabolomics have provided the potentials to detect both oral and systemic diseases. Capillary electrophoresis time-of-flight-mass spectrometry (CE-TOFMS) enables the identification and quantification of various charged metabolites. This method has been employed to biomarker discoveries using human saliva samples, especially for various ...
    Radioactive Assay of in vitro Glutamylation Activity of the Legionella pneumophila Effector Protein SidJ
    Authors:  Alan G. Sulpizio, Jung-Ho Shin, Marena E. Minelli and Yuxin Mao, date: 10/05/2020, view: 451, Q&A: 0
    [Abstract] The Legionella effector protein SidJ has recently been identified to perform polyglutamylation on another Legionella effector, SdeA, ablating SdeA’s activity. SidJ is a kinase-like protein that requires the small eukaryotic protein calmodulin to perform glutamylation. Glutamylation is a relatively uncommon type of ...
    A Novel and Robust Single-cell Trapping Method on Digital Microfluidics
    [Abstract] Due to cell heterogeneity, the differences among individual cells are averaged out in bulk analysis methods, especially in the analysis of primary tumor biopsy samples from patients. To deeply understand the cell-to-cell variation in a primary tumor, single-cell culture and analysis with limited amount of cells are in high demand. Microfluidics ...
    Double Labeling of PDGFR-β and α-SMA in Swine Models of Acute Kidney Injury to Detect Pericyte-to-Myofibroblast Transdifferentation as Early Marker of Fibrosis
    [Abstract] Growing evidences suggest that peritubular capillaries pericytes are the main source of scar-forming myofibroblasts during chronic kidney disease (CKD), as well as early phases of acute kidney injury (AKI). In a swine model of sepsis and I/R (Ischemia Reperfusion) injury-induced AKI we demonstrated that renal pericytes are able to ...
    Fluidigm Based Single-cell Gene Expression Library Preparation from Patient-derived Small Intestinal Organoids
    Authors:  Kohei Suzuki and Ryuichi Okamoto, date: 10/05/2020, view: 381, Q&A: 0
    [Abstract] In this protocol, we describe our methods to isolate crypts from patients' biopsy samples and to culture human intestinal stem cells as it’s called “organoid.” Beyond that, we describe how to dissociate organoids cells into single cells for single-cell analysis as a further application. This protocol should provide investigators sufficient tools ...
    Novel Protein-oligonucleotide Conjugation Method Involving a High-affinity Capture HaloTag
    Author:  Junshi Yazaki, date: 09/20/2020, view: 809, Q&A: 0
    [Abstract] Highly sensitive quantitative protein profiling can play a key role in the early diagnosis of diseases, such as autoimmune diseases and cancer. We developed a modified protein-oligonucleotide conjugation method termed HaloTag-mediated barcoding, for quantifying protein molecules at a higher sensitivity than conventional protein quantification ...

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