Protocol to isolate mature thymic T cell subsets using fluorescence-activated cell sorting for assessing gene expression by RNA-seq and transcription factor binding across the genome by CUT&amp;RUN

Theofilatos, Dimitris; Äijö, Tarmo; Tsagaratou, Ageliki

doi:10.1016/j.xpro.2022.101839

Cited by 4 publications

(7 citation statements)

References 13 publications

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“…Mice were dissected and thymi were isolated and placed immediately in tubes containing ice-cold T cell medium as previously described. 133 , 134 The tubes were placed on ice until the tissue was processed. Each thymus was placed inside a 70 μm cell strainer (BD Falcon, cat no: 352350) inside a 60 mm plate (BD Falcon) and lysed using the pestle of a 1 mL syringe without needle.…”

Section: Methodsmentioning

confidence: 99%

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Deciphering the TET3 interactome in primary thymic developing T cells

Theofilatos,

Ho,

Waitt

et al. 2024

iScience

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Section: Methodsmentioning

confidence: 99%

“…Each thymus was placed inside a 70 μm cell strainer (BD Falcon, cat no: 352350) inside a 60 mm plate (BD Falcon) and lysed using the pestle of a 1 mL syringe without needle. 133 , 134 Cells were measured in a Novocyte cytometer (Acea Agilent) using the NovoExpress software (Agilent).…”

Section: Methodsmentioning

confidence: 99%

Deciphering the TET3 interactome in primary thymic developing T cells

Theofilatos,

Ho,

Waitt

et al. 2024

iScience

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“…In order to understand iNKT cell biology and function, it is critical to perform molecular analysis. To this end, one might need to isolate DNA/RNA and protein for downstream applications such as real-time PCR to evaluate expression of specific genes (Gioulbasani et al, 2020;Pereira et al, 2014) or to perform multiomic analysis such as RNA-seq to evaluate gene expression, ATAC-seq to evaluate chromatin accessibility, whole-genome bisulfite sequencing to assess DNA methylation, or CMS-IP sequencing to assess distribution of 5-hydroxymethylcytosine (Aijo et al, 2022;Engel et al, 2016;Theofilatos et al, 2022;Tsagaratou et al, 2017). Collectively, the past few years' integrative analyses of multiomic approaches have shed light on the transcriptional and epigenetic regulation of iNKT cells (Morgan & Kee, 2022).…”

Section: Identifying Inkt Cell Subsets Based On Surface Marker Expres...mentioning

confidence: 99%

“…Then, we describe the steps for cell fixation and permeabilization for intracellular staining for transcription factors that define distinct iNKT subsets, such as the iNKT cell lineage-specifying transcription factor PLZF (Kovalovsky et al, 2008;Savage et al, 2008) and RORγt, which defines the NKT17 lineage. Based on the expression levels of PLZF and RORγt, we can distinguish NKT1, NKT2, and NKT17 cells (Aijo et al, 2022;Gioulbasani et al, 2020;Tsagaratou et al, 2017) Additional reagents and equipment for mouse euthanasia, organ dissection, and cell counting and for preparing cold cell surface master mix for total iNKT cells (see recipe)…”

Section: Identifying Inkt Cell Subsets Based On Transcription Factor ...mentioning

confidence: 99%

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Defining iNKT Cell Subsets and Their Function by Flow Cytometry

Gioulbasani

Tsagaratou

2023

Current Protocols

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This article discusses methods to assess invariant natural killer T (iNKT) cell subsets isolated from the thymus, as well as the spleen, the liver, and the lung. iNKT cells can be subdivided in distinct, functional subsets based on the transcription factors they express and the cytokines they produce to regulate the immune response. Basic Protocol 1 focuses on characterizing murine iNKT subsets ex vivo by flow cytometry by evaluating the expression of lineagespecifying transcription factors such as PLZF and RORγt. The Alternate Protocol describes a detailed approach to define subsets based on expression of surface markers. This approach can be very useful for maintaining the subsets alive, without fixing them, in order to isolate them for downstream molecular assays such as DNA/RNA isolation, genome-wide analysis to assess gene expression (such as RNA-seq), assessment of chromatin accessibility (for instance, by ATAC-seq), and assessment of DNA methylation by whole-genome bisulfite sequencing. Basic Protocol 2 describes the functional characterization of iNKT cells, which are activated in vitro with PMA and ionomycin for a short period of time and subsequently stained and characterized for production of cytokines, such as IFNγ and IL-4, by flow cytometry. Basic Protocol 3 describes the process of activating iNKT cells in vivo using α-galactosyl-ceramide, a lipid that can be recognized specifically by iNKT cells, allowing assessment of their functionality in vivo. Cells are then isolated and directly stained for cytokine secretion.

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TET proteins regulate Drosha expression and impact microRNAs in iNKT cells

Gioulbasani,

Aijo,

Valenzuela

et al. 2024

Preprint

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DNA demethylases TET2 and TET3 play a fundamental role in thymic invariant natural killer T (iNKT) cell differentiation by mediating DNA demethylation of genes encoding for lineage specifying factors. Paradoxically, differential gene expression analysis revealed that significant number of genes were upregulated upon TET2 and TET3 loss in iNKT cells. This unexpected finding could be potentially explained if loss of TET proteins was reducing the expression of proteins that suppress gene expression. In this study, we discover that TET2 and TET3 synergistically regulate Drosha expression, by generating 5hmC across the gene body and by impacting chromatin accessibility. As DROSHA is involved in microRNA biogenesis, we proceed to investigate the impact of TET2/3 loss on microRNAs in iNKT cells. We report that among the downregulated microRNAs are members of the Let-7 family that downregulate in vivo the expression of the iNKT cell lineage specifying factor PLZF. Our data link TET proteins with microRNA expression and reveal an additional layer of TET mediated regulation of gene expression.

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Protocol to isolate mature thymic T cell subsets using fluorescence-activated cell sorting for assessing gene expression by RNA-seq and transcription factor binding across the genome by CUT&RUN

Cited by 4 publications

References 13 publications

Deciphering the TET3 interactome in primary thymic developing T cells

Deciphering the TET3 interactome in primary thymic developing T cells

Defining iNKT Cell Subsets and Their Function by Flow Cytometry

TET proteins regulate Drosha expression and impact microRNAs in iNKT cells

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