Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

Summary

This study uses flow cytometry and two different gating strategies on isolated perfused mice brain choroid plexuses; this protocol identifies the main immune cell subsets that populate this brain structure.

Abstract

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can indirectly shape brain function. At the interface between the brain and the systemic circulation, the choroid plexuses (CP), which constitute the blood-cerebrospinal fluid barrier, have been highlighted as a key site of periphery-to-brain communication. CP produce the cerebrospinal fluid, neurotrophic factors, and signaling molecules that can shape brain homeostasis. CP are also an active immunological niche. In contrast to the brain parenchyma, which is populated mainly by microglia under physiological conditions, the heterogeneity of CP immune cells recapitulates the diversity found in other peripheral organs. The CP immune cell diversity and activity change with aging, stress, and disease and modulate the activity of the CP epithelium, thereby indirectly shaping brain function. The goal of this protocol is to isolate murine CP and identify about 90% of the main immune subsets that populate them. This method is a tool to characterize CP immune cells and understand their function in orchestrating periphery-to-brain communication. The proposed protocol may help decipher how CP immune cells indirectly modulate brain function in health and across various disease conditions.

Introduction

Since the discovery of the blood-brain barrier by Paul Erhlich in the late 19^th century, the brain has been considered virtually separated from the other organs and the bloodstream. Yet, this last decade has seen the emergence of the concept that brain function is shaped by various biological factors, such as gut microbiota and systemic immune cells and signals^1,2,3,4. In parallel, other brain borders such as meninges and choroid plexuses (CP) have been identified as interfaces of active immune-brain cross talk rather than inert b....

Protocol

All the procedures agreed with the guidelines of the European Commission for the handling of laboratory animals, directive 86/609/EEC. They were approved by the ethical committees No. 59, by the CETEA/CEEA No. 089, under the number dap210067 and APAFIS #32382-2021070917055505 v1.

1. Preparation of the materials

1. Store all antibodies (Table of Materials) at 4 °C, protected from light exposure.
2. DAPI stock solution (1 mg/mL): Resuspend the powder in PBS^-/- (Table of Materials), aliquot, and store at -20 °C.
3. DAPI working solution (0.1 mg/mL): ....

Results

The flow cytometry analyses presented here successfully revealed the major subsets of myeloid and T cells (Figure 1 and Figure 2, respectively), and their relative total number per mouse in a highly reproducible manner (Figure 3).

The flow cytometry analysis of myeloid cells showed that CP are populated by CD11b⁺ CX₃CR1⁺ F4/80^high BAM, representing almost 80% of.......

Discussion

Studies aiming to understand the immunological contributions to brain homeostasis and disease have mainly focused on cells residing within the brain parenchyma, neglecting brain borders such as CP, which are nevertheless crucial contributors to brain function^2,3. The analysis of immune cell populations at CP is challenging due to the small size of CP, low numbers of resident immune cells, and complicated access to this tissue. Flow cytometry performed on total br.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
anti-mouse CD16/CD32	BD Biosciences	553142	Flow cytometry antibody
Albumin, bovine	MP Biomedicals	160069	Blocking reagent
APC anti-mouse CX3CR1	BioLegend	149008	Flow cytometry antibody
APC anti-mouse TCRb	BioLegend	109212	Flow cytometry antibody
APC-Cy7 anti-mouse CD4	BioLegend	100414	Flow cytometry antibody
APC-Cy7 anti-mouse IA-IE	BioLegend	107628	Flow cytometry antibody
BD FACSymphony A5 Cell Analyzer	BD Biosciences		Flow cytometry analyzer
BV711 anti-mouse Ly6C	BioLegend	128037	Flow cytometry antibody
Collagenase IV	Gibco	17104-019	Enzyme to dissociate CP tissue
DAPI	Thermo Scientific	62248	Live/dead marker
EDTA			Ion chelator
fine scissors	FST	14058-11	Dissection tool
FITC anti-mouse CD45	BioLegend	103108	Flow cytometry antibody
Flow controller infusion inset	CareFusion	RG-3-C	Blood perfusion inset
FlowJo software	BD Biosciences		Analysis software
forceps	FST	11018-12	Dissection tool
Heparin	Sigma-Aldrich	H3149-10KU	Anticoagulant
Imalgene	Boehringer Ingelheim		Ketamine, anesthesic
OneComp eBeads	Invitrogen	01-1111-42	Control beads to realize compensation
PBS-/-	Gibco	14190-094	Buffer
PBS+/+	Gibco	14040-091	Buffer
PE anti-mouse CD8a	BioLegend	100708	Flow cytometry antibody
PE anti-mouse F4/80	BioLegend	123110	Flow cytometry antibody
PE-Dazzle 594 anti-mouse CD11b	BioLegend	101256	Flow cytometry antibody
Rompun	Bayer		Xylazine, anesthesic
thin forceps	Dumoxel Biology	11242-40	Dissection tool
Vetergesic	Ceva		Buprenorphin, analgesic