Potterfitzgerald2671

Metabolomics, lipidomics, and the study of cellular metabolism are gaining increasing interest particularly in the field of immunology, since the activation and effector functions of immune cells are profoundly controlled by changes in cellular metabolic asset. Ac-DEVD-CHO mw Among the different techniques that can be used for the evaluation of cellular metabolism, the Seahorse Extracellular Flux Analyzer allows the real time measurement of both glycolytic and mitochondrial respiration pathways in cells of interest, through the assessment of extracellular acidification and oxygen consumption rate. Metabolomics, on the other hand, is the high-throughput analysis of metabolites, i.e., the substrates, intermediates, and products of cellular metabolism, starting from biofluids, cells or tissues. The metabolome does not include lipids as their properties are different from water-soluble metabolites and are classified under the lipidome. Lipidomics analysis allows the identification and quantification of lipid species. Metabolomics and lipidomics are currently performed with mass-spectrometry coupled with liquid or gas chromatography (LC-MS or GC-MS) and/or nuclear-magnetic resonance (NMR). Here we describe the protocol for the evaluation of metabolic rate, metabolomics, and lipidomics in T cells, examining the detailed experimental approaches.The dynamic regulation of protein function by altered protein expression and post-translational modifications (PTMs) is essential for T cell function, but it has remained difficult to systemically quantify such events. Mass spectrometry (MS)-based proteomics has become a mainstream tool for comprehensive profiling of proteome and PTMs, especially with the development of multiplexed isobaric labeling methods, such as tandem mass tag (TMT), coupled with high-resolution two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS). Here, we introduce a deep proteomics profiling protocol with an optimized 11-plex TMT-LC/LC-MS/MS platform to quantitate whole proteome, phosphoproteome, acetylome, and methylome in activated T cells. The major steps include preparation of activated T cells, protein extraction and digestion, TMT labeling, basic pH reverse phase LC, modified peptide enrichment, acidic pH reverse phase LC-MS/MS, and computational data processing. Approximately 10,000 proteins, 30,000 phosphosites, 2,000 lysine acetylated sites, and 1,000 lysine methylated sites can be identified and quantified from 1 mg of proteins per sample. Quality control steps are implemented in this protocol, and future development, such as nanoscale 16-plex TMT analysis, is discussed. This multiplexed and robust method provides a powerful tool for dissecting proteomic and PTM signatures in T cells at the systems level, and it is equally suitable for other biological samples, including effector T cell subsets.During the last decade, the rapid progress in the development of next-generation sequencing (NGS) technologies has provided relevant insights into complex biological systems, ranging from cancer genomics to microbiology. Among NGS technologies, single-cell RNA sequencing is currently used to decipher the complex heterogeneity of several biological samples, including T cells. Even if this technique requires specialized equipment and expertise, nowadays it is broadly applied in research. In this chapter, we will provide an optimized protocol for the isolation of T cells and the preparation of RNA sequencing libraries by using droplet digital technology (ddSEQ, Bio-Rad Laboratories). We will also illustrate a guide to the main steps of data processing and options for data interpretation. This protocol will support users in building a single-cell experimental framework, from sample preparation to data interpretation.The comparison of methylomes from immune cells enables the identification of differentially methylated regions and thereby region-associated gene loci. Those regions can be used to discriminate one immune cell population from the other, as well as help to identify key molecules and major pathways determining the unique phenotypes of immune cell lineages. The combination of bisulfite treatment of genomic DNA and next-generation sequencing provides the basis for studying epigenetic changes in different immune cell populations. Further development of whole-genome bisulfite sequencing resulted in a protocol for sequencing libraries that accept both single- or double-stranded DNA from fixed or nonfixed cells, respectively. Therefore, researchers can include immune cell populations in their methylation studies whose isolation depends on the staining of intracellular molecules.The CRISPR/Cas technology allows for genome editing in primary T cells. We herein describe the activation of primary murine CD4+ or CD8+ T cells, followed by electroporation with plasmid or ribonucleoproteins (RNP) for gene modification. Gene edited T cells can subsequently be transferred to host mice for in vivo studies or cultured in vitro for further characterization. This protocol enables sophisticated genetic analysis of T cells using commonly available virus-free reagents.Lentivirus-mediated gene transfer is an efficient method to introduce a variety of transgenes to human T cells. Here we describe a protocol to transduce human CD4+, CD8+, or CD4+ regulatory T cells. To illustrate the method, we use transduction with lentivirus encoding an HLA-A2-specific chimeric antigen receptor (CAR) and a transduction marker as an example. Methods to isolate, transduce, purify, and expand CD4+ and CD8+ T cells as well as regulatory T cells are provided. We also describe how to carry out cytotoxicity or suppression assays to assess the function of the resulting CAR T cell or CAR regulatory T cells, respectively.Electroporation enables the transfection of different cell types including microbial, plant, and animal cells with charged molecules, such as nuclear acids or proteins. During electroporation, an electrical field is applied to the cells leading to a transient permeabilization of the cell membrane allowing exogenous molecules to enter the cells. Here we report the electroporation of human primary CD4+ -T cells with in-vitro transcribed mRNA to facilitate gene editing (knockout) of the CC-chemokine receptor 5 (CCR5), the coreceptor of the human immunodeficiency virus 1 (HIV1) predominantly used during primary infection. Using such strategy of transient expression of a CCR5-specific Transcription-activator-like-effector nuclease (TALEN), we aim to protect helper T cells from de novo HIV infection.