Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

Summary

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Abstract

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

Introduction

Though the gastrointestinal (GI) tract is primarily dedicated to the processing and reabsorption of nutrients from food, the GI tract also maintains central roles in the integrity of the vascular, lymphatic, and nervous systems and of numerous other organs through its mucosal and submucosal immune system¹. The GI immune system has an influential role in both gastrointestinal and systemic health due to its constant exposure to foreign antigens from food, commensal bacteria, or invading pathogens^1,2. Thus, the GI immune system must maintain a delicate balance in which it tolerates non-pat....

Protocol

All studies were conducted under rodent research protocols reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Miami Miller School of Medicine, which meets the veterinary standards set by the American Association for Laboratory Animal Science (AALAS).

1. Preparation of Solutions

1. As described in Table 1, prepare the Colon Buffer, Silica-Based Density Separation Media 100%, Silica-Based Density Separation Media 66%, Silica-based Density Separation Media 44%, Collagenase E Digestion Buffer, and FACS Buffer.
   1. Prepare Colon Buffer the day prior to the proce....

Results

When working with murine colon disease models, it is helpful to be able to both quantify and qualitatively assess, among the MNC of the colon, multiple immune cell subsets involved in the inflammatory process. The single-cell suspension of MNC obtained through the application of this protocol facilitates such phenotypic characterization in a robust and reproducible manner. As a proof of principle for the application of this isolation method under diverse experimental settings, we retrieve.......

Discussion

This visual protocol describes well-tolerated methods for the isolation of colonic mononuclear cells including lamina propria lymphocytes (LPL). Given that this protocol was optimized in evaluating severe post-transplant mouse colitis models where inflammatory cytokines and tissue injury lend themselves to poor viability of recovered MNC, we anticipate that these methods can be translated to other applications requiring phenotypic analysis of colonic MNC. These include but, are not limited to assessing colon inflammation.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
60 mm Petri DIsh	Thermo Scientific	150288
1x PBS	Corning	21-040-CV
10x PBS	Lonza BioWhittaker	BW17-517Q
10 mL Disposable Serological Pipette	Corning	4100
10 mL Syringe	Becton Dickinson	302995
15 mL Non-Sterile Conical Tubes	TruLine	TR2002
18 G Blunt Needle	Becton Dickinson	305180
25 mL Disposable Serological Pipette	Corning	4250
40 μm pore size Cell Strainer	Corning	352340
50 mL Falcon Tube	Corning	21008-951
Bovine Serum Albumin (BSA)	Sigma	A4503-1KG
Fixation Buffer	Biolegend	420801
E. coli Collagenase E from Clostridium histolyticum	Sigma	C2139
EDTA, 0.5 M Sterile Solution	Amresco	E177-500ML
Fetal Bovine Serum	Thermo /Fisher Scientific -HyCLone	SV30014.03
HEPES	GE Healthcare-HyClone	SH30237.01
Percoll	GE Healthcare-Life Sciences	1708901
RPMI Medium	Corning	17-105-CV
Sodium Azide	VWR Life Science Amresco	97064-646
Trypan Blue	Lonza BioWhittaker	17-942E