Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Summary

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Abstract

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria ¹. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year ², and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse. 

Protocol

1. Removal of the Genital Tract

1. Euthanize the mouse using approved guidelines. Make low midline incision and retract skin.
2. Isolate and remove whole genital tract, from oviduct to vaginal opening.
3. Open the entire tract longitudinally with scissors.
4. Cut the genital tract with fine surgical scissors into fine pieces (about <1 mm) in a Petri dish with Complete RPMI-10/EDTA (see the formula below) and stir the tissue in 125 ml flask at room temperature on a magnetic stirrer set for 15 min. This procedure is repeated twice and then the supernatant, which contains epithelium and other debris, is discarded.
5. Pour contents of ....

Discussion

In this protocol, it is shown how to prepare lymphocytes from mouse genital tracts and is easily mastered. The most critical point of this process is to maintain cell viability by keeping the cells on ice. The cell yield from this method depends on technique, handling skill, the infectious status of the mouse (naÏve or day after infection), the pathogen (bacterial, viral, fungal) and the section of the murine reproductive tract examined (oviduct, uterus, vaginal segment or entire genital tract). Our group and others.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Collagenase	Sigma Life Sciences	C2139-1G
Percoll	GE Healthcare Bio-Sciences	17-0891-01
RPMI1640	Gibco by Life Technologies	11875