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Immunology

Categories
+ Animal model
+ Antibody analysis
+ Complement analysis
+ Host defense
+ Immune cell differentiation
+ Immune cell function
+ Immune cell imaging
+ Immune cell isolation
+ Immune cell staining
+ Immune mechanisms
+ Immunotherapy
+ Inflammatory disorder
- Mucosal immunology
Digestive tract
Epithelium
+ Genitourinary tract
Vaccine adjuvant
Protocols in Past Issues

Standardized Flow Cytometry Method for Absolute Counting of Intraepithelial Lymphocytes in the Intestinal Mucosa Using TruCountTM Beads
CJ Corentin Joulain
SB Stéphanie Bessoles
AC Andrada S. Chiron
GS Guillaume Sarrabayrouse
SH Salima Hacein-Bey-Abina
Q&A 0 Q&A
2257 Views
May 5, 2025

In the intestinal epithelium, intraepithelial lymphocytes (IELs) coexist with intestinal epithelial cells (IECs). The IELs have an important role in defending the intestinal tract against pathogens and eliminating tumor cells. Anomalies in the absolute IEL count have been reported in various digestive diseases. IELs are typically counted using histologic techniques or under light microscopy after isolation of the epithelium. However, these techniques can introduce bias, which might account for the discrepancies in counts from one study to another. Here, we describe a flow cytometry assay for determining the absolute IEL count and the IEL/IEC ratio. We combined a conventional epithelial isolation method with a BD TruCountTM bead-based absolute counting technique to quantify IELs (CD45+ CD326/EpCAM- CD103+CD3+) and IECs (CD45- CD326/EpCAM+) in a C57BL/6 mouse model.

Intravital Imaging of Intestinal Intraepithelial Lymphocytes
SM Sara McArdle
GS Goo-Young Seo
MK Mitchell Kronenberg
ZM Zbigniew Mikulski
Q&A 0 Q&A
2575 Views
Jul 20, 2023

Intestinal intraepithelial lymphocytes (IEL) are a numerous population of T cells located within the epithelium of the small and large intestines, being more numerous in the small intestine (SI). They surveil this tissue by interacting with epithelial cells. Intravital microscopy is an important tool for visualizing the patrolling activity of IEL in the SI of live mice. Most IEL express CD8α; therefore, here we describe an established protocol ofintravital imaging that tracks lymphocytes labeled with a CD8α-specific monoclonal antibody in the SI epithelium of live mice. We also describe data acquisition and quantification of the movement metrics, including mean speed, track length, displacement length, and paths for each CD8α+ IEL using the available software. The intravital imaging technique for measuring IEL movement will provide a better understanding of the role of IEL in homeostasis and protection from injury or infection in vivo.

Method for Measuring Mucociliary Clearance and Cilia-generated Flow in Mice by ex vivo Imaging
ES Eric Song
AI Akiko Iwasaki
Q&A 0 Q&A
5024 Views
Mar 20, 2020
Ex vivo biophysical measurements provide valuable insights into understanding both physiological and pathogenic processes. One critical physiological mechanism that is regulated by these biophysical properties is cilia-generated flow that mediates mucociliary clearance, which is known to provide protection against foreign particles and pathogens in the upper airway. To measure ciliary clearance, several techniques have been implemented, including the use of radiolabeled particles and imaging with single-photon emission computerized tomography (SPECT) methods. Although non-invasive, these tests require the use of specialized equipment, limiting widespread use. Here we describe a method of ex vivo imaging of cilia-generated flow, adapted from previously reported methods, to make it more accessible and higher throughput for researchers. We excise trachea from mice quickly after euthanasia, cut it longitudinally and place it in an inhouse made slide. We apply fluorescent particles to measure particle movement under a fluorescent microscope, followed by analysis with ImageJ, allowing calculation of fluid flow generated by cilia under different conditions. This method enables ex vivo measurements in tissue with minimal investment or special equipment, giving opportunity to investigate and discover important biophysical properties associated with ciliary movement of the trachea in physiology and disease.

Eimeria vermiformis Infection Model of Murine Small Intestine
PF Patrícia Figueiredo-Campos
CF Cristina Ferreira
BB Birte Blankenhaus
MV Marc Veldhoen
Q&A 0 Q&A
6688 Views
Dec 20, 2018
Eimeria vermiformis is a tissue specific, intracellular protozoan that infects the murine small intestinal epithelia, which has been widely used as a coccidian model to study mucosal immunology. This mouse infection model is valuable to investigate the mechanisms of host protection against primary and secondary infection in the small intestine. Here, we describe the generation of an E. vermiformis stock solution, preparation of sporulated E. vermiformis to infect mice and determination of oocysts burden. This protocol should help to establish a highly reproducible natural infection challenge model to study immunity in the small intestine. The information obtained from using this mouse model can reveal fundamental mechanisms of interaction between the pathogen and the immune response, e.g., provided by intraepithelial lymphocytes (IEL) at the basolateral site of epithelial cells but also a variety of other immune cell populations present in the gut.

Adhesion of Enteroaggregative E. coli Strains to HEK293 Cells
JA Jorge Luis Ayala-Lujan
FR Fernando Ruiz-Perez
Q&A 0 Q&A
6889 Views
Apr 20, 2018
Enteroaggregative Escherichia coli (EAEC) is a recognized cause of acute diarrhea among both children and adults worldwide. EAEC strains are characterized by the presence of aggregative adherence fimbriae (AAF), which play a key role in pathogenesis by mediating attachment to the intestinal mucosa and by triggering host inflammatory responses. The aggregative adherence fimbria II (AAF/II) is the most important adherence factor of EAEC prototype strain 042 (EAEC042) to intestinal cells. Multiple receptors for AAF/II on epithelial cells have been identified including the transmembrane signaling mucin Muc1. This protocol describes a method to measure adherence of EAEC strains to HEK293 cells expressing the Muc1 glycoprotein.

Immunofluorescence Analysis of Human Endocervical Tissue Explants Infected with Neisseria gonorrhoeae
LW Liang-Chun Wang
QY Qian Yu
DS Daniel C. Stein
WS Wenxia Song
Q&A 0 Q&A
7980 Views
Feb 5, 2018
Colonization and penetration of the epithelium is the infection-initiating route of mucosal pathogens. The epithelium counteracts infection by eliciting host cell responses while maintaining the mucosal barrier function. The obligate human sexually transmitted bacterium Neisseria gonorrhoeae, or gonococcus (GC) infects the female reproductive tract primarily from the endocervical epithelium. Due to lack of an infection model that mimics all aspects of human infections in the female reproductive tract, GC pathogenesis is poorly understood. This protocol takes advantage of the viability and functional integrity of human cervical tissues propagated in culture to generate an ex vivo infection model. This tissue model maintains the nature of the infection target and environment without any manipulation such as immortalization of epithelial cells by viruses. Using immunofluorescence microscopy, the interaction of GC with the endocervical epithelium was analyzed.

Study of Epithelium Barrier Functions by Real-time TER Measurement
JM Jingjing Meng
SR Sabita Roy
Q&A 0 Q&A
8762 Views
May 20, 2016
Transepithelial Electrical Resistance (TER) measurement is a reliable and efficient method to quantify the permeability of barrier forming cells such as epithelial cells. Measuring the permeability of the epithelial cells will help the researchers to investigate the barrier function of epithelium in various infectious and inflammatory diseases. Here we provide a real-time and impedance-based approach for measuring the permeability of epithelial cell monolayer using the Electrical Cell Substrate Impedance Sensing (ECIS®) instrumentation.