![]() These observations raise the interesting possibility that the NL helps to establish a repressive environment. When cells differentiate, detachment of genes from the NL often coincides with transcriptional activation, while increased NL interactions correlate with reduced transcription (Peric-Hupkes et al, 2010 Lund et al, 2013 Robson et al, 2016, 2017). Most genes inside LADs have very low transcriptional activity (Guelen et al, 2008 Peric-Hupkes et al, 2010 Leemans et al, 2019). How LAD-NL contacts are regulated is poorly understood. The NL contacts of some LADs are highly consistent between cell types, while other LADs interact in cell-type-specific (facultative) manners with the NL. Mammalian genomes have roughly one thousand of such lamina-associated domains (LADs), which are typically hundreds of kb or even a few Mb in size. In metazoan cell nuclei, large chromatin domains are associated with the nuclear lamina (NL) (Gonzalez-Sandoval & Gasser, 2016 van Steensel & Belmont, 2017 de Leeuw et al, 2018 Kim et al, 2019 Lochs et al, 2019). These extensive datasets are a resource for the analysis of LAD rewiring by transcription and reveal a remarkable flexibility of interphase chromosomes. Inactivation of active genes can lead to increased NL contacts. Loss of NL interactions coincides with a switch from late to early replication timing, but the latter can involve longer stretches of DNA. ![]() The degree of detachment depends on the expression level and the length of the activated gene. Gene activation inside LADs typically causes NL detachment of the entire transcription unit, but rarely more than 50–100 kb of flanking DNA, even when multiple neighboring genes are activated. We addressed these questions by systematic activation or inactivation of individual genes, followed by detailed genome-wide analysis of NL interactions, replication timing, and transcription patterns. How this process works and how it impacts flanking chromosomal regions are poorly understood. Activation of such genes is often accompanied by repositioning toward the nuclear interior. Our approach provides robust protocols for affordable, semi-automated eight-color cytometric immunophenotyping that can be used in population-based studies and clinical trial settings.Transcriptionally inactive genes are often positioned at the nuclear lamina (NL), as part of large lamina-associated domains (LADs). This work establishes a foundation for defining reference values in healthy donors. We report on four panels that were designed to enumerate and phenotype major immune cell populations (PMN, T, B, NK cells, monocytes and DC). We optimized eight-color antibody panels and procedures for staining and lysis of whole blood samples, and implemented pre-analytic steps with a semi-automated workflow using a robotic system. Herein we report the approach taken by the Milieu Intérieur Consortium, highlighting the standardized and automated procedures used for immunophenotyping of human whole blood samples. Standardized procedures are essential to allow for inter-individual comparisons in the context of population based or clinical studies. Immunophenotyping by multi-parametric flow cytometry is the cornerstone technology for enumeration and characterization of immune cell populations in health and disease.
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