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.
Biochemical methods can reveal stable protein‐protein interactions occurring within cells, but the ability to observe transient events and to visualize the subcellular localization of protein‐protein interactions in cells and tissues in situ provides important additional information. The Proximity Ligation Assay®(PLA) offers the opportunity to visualize the subcellular location of such interactions at endogenous protein levels, provided that the probes that recognize the target proteins are within 40 nm. This sensitive technique not only elucidates protein‐protein interactions, but also can reveal post‐translational protein modifications. The technique is useful even in cases where material is limited, such as when paraffin‐embedded clinical specimens are the only available material, as well as after experimental intervention in 2D and 3D model systems. Here we describe the basic protocol for using the commercially available Proximity Ligation Assay™ materials (Sigma‐Aldrich, St. Louis, MO), and incorporate details to aid the researcher in successfully performing the experiments. © 2020 Wiley Periodicals LLC.
- PAR ID:
- 10236010
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols in Cell Biology
- Volume:
- 89
- Issue:
- 1
- ISSN:
- 1934-2500
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract 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 -
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Abstract Protein labeling strategies have been explored for decades to study protein structure, function, and regulation. Fluorescent labeling of a protein enables the study of protein‐protein interactions through biophysical methods such as microscale thermophoresis (MST). MST measures the directed motion of a fluorescently labeled protein in response to microscopic temperature gradients, and the protein's thermal mobility can be used to determine binding affinity. However, the stoichiometry and site specificity of fluorescent labeling are hard to control, and heterogeneous labeling can generate inaccuracies in binding measurements. Here, we describe an easy‐to‐apply protocol for high‐stoichiometric, site‐specific labeling of a protein at its N‐terminus with
N ‐hydroxysuccinimide (NHS) esters as a means to measure protein‐protein interaction affinity by MST. This protocol includes guidelines for NHS ester labeling, fluorescent‐labeled protein purification, and MST measurement using a labeled protein. As an example of the entire workflow, we additionally provide a protocol for labeling a ubiquitin E3 enzyme and testing ubiquitin E2‐E3 enzyme binding affinity. These methods are highly adaptable and can be extended for protein interaction studies in various biological and biochemical circumstances. © 2021 Wiley Periodicals LLC.This article was corrected on 18 July 2022. See the end of the full text for details.
Basic Protocol 1 : Labeling a protein of interest at its N‐terminus with NHS esters through stepwise reactionAlternate Protocol : Labeling a protein of interest at its N‐terminus with NHS esters through a one‐pot reactionBasic Protocol 2 : Purifying the N‐terminal fluorescent‐labeled protein and determining its concentration and labeling efficiencyBasic Protocol 3 : Using MST to determine the binding affinity of an N‐terminal fluorescent‐labeled protein to a binding partner.Basic Protocol 4 : NHS ester labeling of ubiquitin E3 ligase WWP2 and measurement of the binding affinity between WWP2 and an E2 conjugating enzyme by the MST binding assay -
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Abstract Protein tyrosine
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Abstract Long read sequencing technologies now allow high‐quality sequencing of RNAs (or their cDNAs) that are hundreds to thousands of nucleotides long. Long read sequences of nascent RNA provide single‐nucleotide‐resolution information about co‐transcriptional RNA processing events—e.g., splicing, folding, and base modifications. Here, we describe how to isolate nascent RNA from mammalian cells through subcellular fractionation of chromatin‐associated RNA, as well as how to deplete poly(A)+RNA and rRNA, and, finally, how to generate a full‐length cDNA library for use on long read sequencing platforms. This approach allows for an understanding of coordinated splicing status across multi‐intron transcripts by revealing patterns of splicing or other RNA processing events that cannot be gained from traditional short read RNA sequencing. © 2020 Wiley Periodicals LLC.
Basic Protocol 1 : Subcellular fractionationBasic Protocol 2 : Nascent RNA isolation and adapter ligationBasic Protocol 3 : cDNA amplicon preparation -
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Abstract Three‐dimensional cell encapsulation has rendered itself a staple in the tissue engineering field. Using recombinantly engineered, biopolymer‐based hydrogels to encapsulate cells is especially promising due to the enhanced control and tunability it affords. Here, we describe in detail the synthesis of our hyaluronan (i.e., hyaluronic acid) and elastin‐like protein (HELP) hydrogel system. In addition to validating the efficacy of our synthetic process, we also demonstrate the modularity of the HELP system. Finally, we show that cells can be encapsulated within HELP gels over a range of stiffnesses, exhibit strong viability, and respond to stiffness cues. © 2023 Wiley Periodicals LLC.
Basic Protocol 1 : Elastin‐like protein modification with hydrazineBasic Protocol 2 : Nuclear magnetic resonance quantification of elastin‐like protein modification with hydrazineBasic Protocol 3 : Hyaluronic acid–benzaldehyde synthesisBasic Protocol 4 : Nuclear magnetic resonance quantification of hyaluronic acid–benzaldehydeBasic Protocol 5 : 3D cell encapsulation in hyaluronan elastin‐like protein gels
