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
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.
1. Removal of the Genital Tract
2. Digesting Genital Tract Tissue
3. Separating Genital Tract Cells Using Percoll Gradients
4. Representative Results
An example of isolation of lymphocytes from mouse genital tract and flow cytometry (FACS) analysis is shown in Figure 1. Genital tract was removed from Chlamydia muridarum intravaginally infected mouse 7 days after infection. Two genital tracts were pooled together in order to have enough lymphocytes for functional and phenotype examination by FACS. Dissected tissues were processed by the digestion and isolation steps as outlined above. The single cell suspension was stained for various fluorochrome-conjugated monoclonal antibodies against mouse CD3, CD4 and CD8. Figure 1 shows a dot-plot presentation of CD4 positive T cells versus CD8 positive T cells gated on CD3 positive T lymphocytes. Our procedure can examine lymphocytes isolated from genital tracts of different disease models to assess various immune cell functional and phenotypic characteristics in different diseases in mouse model. These include diverse immune cells, such as dendritic cells, neutrophil, macrophages NK, NKT, T cells (Ag-specific T cells), B cells etc.3-10.
Figure 1. Lymphocytes were isolated from genital tracts of mice infected with Chlamydia muridarum, using the method presented here. After the complete separation procedure, lymphocytes were stained with fluorochrome-conjugated CD3, CD4 and CD8. The quadrant data were gated on CD3+ lymphocytes, showing CD4+ versus CD8+ with and percentage of gated cells.
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 have published various studies on different lymphocyte populations from mouse genital tract 5-7. On our hands, from one naÏve BALB/c mouse, entire genital tract can generate 2×106 cells with 90% viability. In many occasions, the lymphocytes will be used for further functional and/or flow cytometric analysis 3-10. In comparison with the commonly used isolation method 11, by our method, the cell yield is increased 10 times. This procedure is relatively long, but it can be simplified depending on the further use of the isolated lymphocytes. When the cells are used for non-functional assays such as flow cytometry analysis, the method can be shorted by skipping Percoll gradient step. This method provides a powerful and an efficient tool to study mucosal lymphocytes and provide insight into infectious disease research, facilitate subsequent vaccine development.
The authors have nothing to disclose.
We like to thank NIH for funding grants R01-AI026328 (KAK) and R01-AI079004 (KAK).
Name | Company | Catalogue number | |
Collagenase | Sigma Life Sciences | C2139-1G | |
Percoll | GE Healthcare Bio-Sciences | 17-0891-01 | |
RPMI1640 | Gibco by Life Technologies | 11875 |