In this invited article, we explain technical aspects of the lymphocytic choriomeningitis virus (LCMV) system, providing an update of a prior contribution by Matthias von Herrath and J. Lindsay Whitton. We provide an explanation of the LCMV infection models, highlighting the importance of selecting an appropriate route and viral strain. We also describe how to quantify virus‐specific immune responses, followed by an explanation of useful transgenic systems. Specifically, our article will focus on the following protocols. © 2020 Wiley Periodicals LLC.
Drug‐induced liver injury is an important cause of non‐approval in drug development and the withdrawal of already approved drugs from the market. Screening human hepatic cell lines for toxicity has been used extensively to predict drug‐induced liver injury in preclinical drug development. Assessing hepatic‐cell health with more diverse markers will increase the value of in vitro assays and help predict the mechanism of toxicity. We describe three live cell‐based assays using HepG2 cells to measure cell health parameters indicative of hepatotoxicity. The first assay measures cellular ATP levels using luciferase. The second and third assays are multiparametric high‐content screens covering a panel of cell health markers including cell count, mitochondrial membrane potential and structure, nuclear morphology, vacuolar density, and reactive oxygen species and glutathione levels. © 2020 Wiley Periodicals LLC.
- PAR ID:
- 10236015
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols in Chemical Biology
- Volume:
- 12
- Issue:
- 4
- ISSN:
- 2160-4762
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Our understanding of programmed cell death 1 (PD‐1) biology is limited due to technical difficulties in establishing reproducible, yet simple, in vitro assays to study PD‐1 signaling in primary human T cells. The protocols in this article were refined to test the consequences of PD‐1 ligation on short‐term T cell signaling, long‐term T cell function, and the structural consequences of PD‐1 ligation with PD‐1 ligands. Basic Protocol 1 addresses the need for a robust and reproducible short‐term assay to examine the signaling cascade triggered by PD‐1. We describe a phospho flow cytometry method to determine how PD‐1 ligation alters the level of CD3ζ phosphorylation on Tyr142, which can be easily applied to other proximal signaling proteins. Basic Protocol 2 describes a plate‐bound assay that is useful to examine the long‐term consequences of PD‐1 ligation such as cytokine production and T cell proliferation. Complementary to that, Basic Protocol 3 describes an in vitro superantigen‐based assay to evaluate T cell responses to therapeutic agents targeting the PD‐1/PD‐L axis, as well as immune synapse formation in the presence of PD‐1 engagement. Finally, in Basic Protocol 4 we outline a tetramer‐based method useful to interrogate the quality of PD‐1/PD‐L interactions. These protocols can be easily adapted for mouse studies and other inhibitory receptors. They provide a valuable resource to investigate PD‐1 signaling in T cells and the functional consequences of various PD‐1‐based therapeutics on T cell responses. © 2020 Wiley Periodicals LLC.
Basic Protocol 1 : PD‐1 crosslinking assay to determine CD3ζ phosphorylation in primary human T cellsBasic Protocol 2 : Plate‐based ligand binding assay to study PD‐1 function in human T cellsSupport Protocol 1 : T cell proliferation assay in the presence of PD‐1 ligationBasic Protocol 3 : In vitro APC/T cell co‐culture system to evaluate therapeutic interventions targeting the PD‐1/PD‐L1 axisSupport Protocol 2 : Microscopy‐based approach to evaluate the consequences of PD‐1 ligation on immune synapse formationBasic Protocol 4 : Tetramer‐based approach to study PD‐1/PD‐L1 interactions -
Abstract The reversible oxidation of protein tyrosine phosphatases (PTPs) impairs their ability to dephosphorylate substrates in vivo. This transient inactivation of PTPs occurs as their conserved catalytic cysteine residue reacts with cellular oxidants thereby abolishing the ability of this reactive cysteine to attack the phosphate of the target substrate. Hence, in vivo, the inhibition of specific PTPs in response to regulated and localized rises in cellular oxidants enables phospho‐dependent signaling. We present assays that measure the endogenous activity of specific PTPs that become transiently inactivated in cells exposed to growth factors. Here, we describe the methods and highlight the pitfalls to avoid post‐lysis oxidation of PTPs in order to assess the inactivation and the reactivation of PTPs targeted by cellular oxidants in signal transduction. © 2020 Wiley Periodicals LLC.
Basic Protocol 1 : Cell transfection (optional)Support Protocol : Preparation of degassed lysis buffersBasic Protocol 2 : Cellular extraction in anaerobic conditionsBasic Protocol 3 : Enrichment and activity assay of specific PTPsAlternate Protocol : Measurement of active PTPs via direct cysteinyl labeling -
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This article was corrected on 18 July 2022. See the end of the full text for details.
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