Université Paris Descartes, Sorbonne Paris Cité

7 ARTICLES PUBLISHED IN JoVE

Immunology and Infection

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Hélène Salmon^1,2, Ana Rivas-Caicedo^1,2, François Asperti-Boursin^1,2, Camille Lebugle^1,2, Pierre Bourdoncle^1,2, Emmanuel Donnadieu^1,2

¹Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), ²Inserm, U1016, Paris, France

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Immunology and Infection

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Florence Marie-Anaïs¹, Julie Mazzolini¹, Pierre Bourdoncle¹, Florence Niedergang¹

¹Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

Immunology and Infection

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Gabrielle Lê-Bury¹, Chantal Deschamps¹, Audrey Dumas¹, Florence Niedergang¹

¹Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

Neuroscience

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

Julie Mazzolini¹, Kelda Chia¹, Dirk Sieger¹

¹Centre for Discovery Brain Sciences, University of Edinburgh

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

Neuroscience

Long-term Sensory Conflict in Freely Behaving Mice

Filipa França de Barros^1,2, Julie Carcaud², Mathieu Beraneck^1,2

¹Integrative Neuroscience and Cognition Center, UMR8002, CNRS, ²Sorbonne Paris Cité, Université Paris Descartes

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Neuroscience

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Louis Richevaux^1,2, Louise Schenberg^1,2, Mathieu Beraneck^1,2, Desdemona Fricker^1,2

¹CNRS (Integrative Neuroscience and Cognition Center, UMR 8002), ²Université Paris Descartes, Sorbonne Paris Cité

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

Immunology and Infection

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Nikaïa Smith^1,2,3,4, Nassima Bekaddour^2,3,4, Nicolas Leboulanger^4,5, Yolande Richard^*6, Jean-Philippe Herbeuval^*2,3,4

¹Institute of Molecular Virology, Ulm University Medical Center, ²CNRS UMR-8601, Centre Interdisciplinaire Chimie Biologie, ³Team Chemistry & Biology, Modeling & Immunology for Therapy, Université Paris Descartes, ⁴Université Paris Descartes, Sorbonne Paris Cité, ⁵Pediatric Otolaryngology Department, Hôpital Necker-Enfants Malades, Assistance Publique Hôpitaux de Paris, ⁶Université de Paris, Institut Cochin

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.