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Production and Bioassay of a Diffusible Factor that Induces Gametophyte-to-Sporophyte Developmental Reprogramming in the Brown Alga Ectocarpus

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Measuring Cell Growth and Junction Development in Epithelial Cells Using Electric Cell-Substrate Impedance Sensing (ECIS)

Authors: Shaista Anwer and Katalin Szaszi, date: 08/20/2020, view: 947, Q&A: 0

[Abstract] Electric Cell-substrate Impedance Sensing (ECIS) is an automated method that can be used to quantify processes such as cell attachment, growth, migration and barrier functions (i.e., the properties of tight junctions). The method provides simultaneous information on cell number and tight junction function by detecting electric parameters ...

SMART (Single Molecule Analysis of Resection Tracks) Technique for Assessing DNA end-Resection in Response to DNA Damage

Authors: Angela Altieri, Milena Dell’Aquila, Francesca Pentimalli, Antonio Giordano and Alfano Luigi, date: 08/05/2020, view: 757, Q&A: 0

[Abstract] DNA double strand breaks (DSBs) are among the most toxic lesions affecting genome integrity. DSBs are mainly repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). A crucial step of the HR process is the generation, through DNA end-resection, of a long 3′ single-strand DNA stretch, necessary to prime DNA synthesis ...

A CRISPR Competition Assay to Identify Cancer Genetic Dependencies

Authors: Vishruth Girish and Jason M. Sheltzer, date: 07/20/2020, view: 1187, Q&A: 0

[Abstract] The CRISPR/Cas9 system is a powerful tool for genome editing, wherein the RNA-guided nuclease Cas9 can be directed to introduce double-stranded breaks (DSBs) at a targeted locus. In mammalian cells, these DSBs are typically repaired through error-prone processes, resulting in insertions or deletions (indels) at the targeted locus. Researchers can ...

Immunofluorescent Staining of Claudin-2 in Cultured Kidney Tubular Cells

Authors: Shaista Anwer and Katalin Szaszi, date: 07/20/2020, view: 483, Q&A: 0

[Abstract] Members of the claudin family of tight junction proteins regulate paracellular permeability and modulate cell signaling. During junction remodeling, these proteins are selectively inserted into or retrieved from the tight junctions, but the control and coordination of these processes remain incompletely understood. Visualization of claudins allows ...

Tyramide Signal-Amplified Immunofluorescence of MYCN and MYC in Human Tissue Specimens and Cell Line Cultures

Authors: Johanna M. Schafer and Jennifer A. Pietenpol, date: 07/05/2020, view: 697, Q&A: 0

[Abstract] MYC family members, MYC, MYCN, and MYCL, are oncogenic transcription factors that regulate the expression of genes involved in normal development, cell growth, proliferation, metabolism, and survival. While MYC is amplified and/or overexpressed across a variety of tissue types, MYCN is often overexpressed in tumors of the nervous system ...

Generation and Testing of Fluorescent Adaptable Simple Theranostic (FAST) Proteins

Authors: Andrew S. Flies, Jocelyn M. Darby, Peter R. Murphy, Terry L. Pinfold, Amanda L. Patchett and Patrick R. Lennard, date: 07/05/2020, view: 1262, Q&A: 0

[Abstract] This protocol provides a step-by-step method to create recombinant fluorescent fusion proteins that can be secreted from mammalian cell lines. This builds on many other recombinant protein and fluorescent protein techniques, but is among the first to harness fluorescent fusion proteins secreted directly into cell culture supernatant. This opens ...

NMR waterLOGSY as An Assay in Drug Development Programmes for Detecting Protein-Ligand Interactions–NMR waterLOGSY

Authors: Carole J. R. Bataille, Terence H. Rabbitts and Timothy D. W. Claridge, date: 07/05/2020, view: 812, Q&A: 0

[Abstract] In drug development programmes, multiple assays are needed for the determination of protein-compound interactions and evaluation of potential use in assays with protein-protein interactions. In this protocol we describe the waterLOGSY NMR method for confirming protein-ligand binding events.

Isolation of Lipid Rafts from Cultured Mammalian Cells and Their Lipidomics Analysis

Authors: Nigora Mukhamedova, Kevin Huynh, Hann Low, Peter J. Meikle and Dmitri Sviridov, date: 07/05/2020, view: 846, Q&A: 0

[Abstract] Lipid rafts are distinct liquid-ordered domains of plasma membranes of most eukaryotic cells providing platform for signaling pathways. Lipid composition of rafts is critical for their structural integrity and for regulation of signaling pathways originating from rafts. Here we provide a protocol to isolate lipid rafts from cultured human and ...

Modeling NOTCH1 driven T-cell Acute Lymphoblastic Leukemia in Mice

Authors: Agnieszka A. Wendorff and Adolfo A. Ferrando, date: 05/20/2020, view: 1222, Q&A: 0

[Abstract] T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that arises from transformation of T-cell primed hematopoietic progenitors. Although T-ALL is a heterogenous and molecularly complex disease, more than 65% of T-ALL patients carry activating mutations in the NOTCH1 gene. The majority of T-ALL–associated ...

Isolation of Tumor Cells Based on Their Distance from Blood Vessels

Authors: Saran Kumar, Husni Sharife, Tirzah Kreisel, Libat Bar-Lev, Myriam Grunewald and Eli Keshet, date: 05/20/2020, view: 1354, Q&A: 0

[Abstract] Differential exposure of tumor cells to microenvironmental cues greatly impacts cell phenotypes, raising a need for position based sorting of tumor cells amenable to multiple OMICs and functional analyses. One such key determinant of tumor heterogeneity in solid tumors is its vasculature. Proximity to blood vessels (BVs) profoundly affects tumor ...

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