Abstract
The lumen of gastrointestinal tract is exposed to several potentially pathogenic microorganisms, thus it is extremely relevant to understand how immunosurveilance can be established. Peyer’s Patches (PPs) are oval or round lymphoid nodules that protrude from the outer wall of the ileum portion of small intestine. PPs contain a high percentage of B and T lymphocytes, macrophages and dendritic cells. Here we summarize a protocol for isolation and culture of mouse PP cells, which can be used to get a better insight into immunopathologies of microbes and to evaluate immune responses elicited by mucosal vaccines.
Keywords: Mucosae, Peyer's Patch, Intestine
Materials and Reagents
Equipment
Procedure
Figure 1. Diagram for the isolation of PP cells from small intestine. Modified from Lefrancois and Lycke (2001).
Representative data
FACS analysis of in vitro activated PP cells. PPs were obtained from 6 Balb/c mice as previously published (Pastori et al., 2014). In detail, two different immunization protocols were used: 3 mice received one intranasal (i. n.), while the other 3 mice received one intraperitoneal (i. p.) immunization. Aluminium hydroxide was used as adjuvant in i. p. immunization. For immunization Flock House Virus (FHV) was used in which an external loop of CCR5 (a seven transmembrane protein, belonging to chemokine receptor family and coreceptor for HIV-1) was introduced. The detailed immunization protocol has been described in Pastori et al. (2014). After excision, PPs were mechanically dissociated and PP cells were in vitro activated with PMA and Ionomycin, as describe above. After 24 h cells were harvested and stained with antibodies against some markers of B cell activation. As shown in Figure 2, CD138 and CD40/CD19 expression indicate a B cell activated phenotype for both protocols. Figure 2. Expression of CD19, CD40, CD138 and co-expression of CD19/CD40 on PP cells after 24 h of PMA/Ionomycin activation. Percentage of positive cells refers to lymphocytes (gated on FSC vs SSC plot). Mean and standard deviation are shown.
Notes
Recipes
Note: Prepare and keep all solutions in a sterile condition.
Acknowledgments
We thank Maria Rescigno for her help in mouse PPs isolation. This work was supported by Italian Ministry of Health, grant 40H15.
References
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