Lamina Propria Mononuclear Cell Isolation: A Silica-based Density Gradient Centrifugation Technique to Obtain Mononuclear Cells from Murine Colon

Abstract

Source: McManus, D. et al. Isolation of lamina propria mononuclear cells from murine colon using collagenase E. J. Vis. Exp. (2019)

In this video, we demonstrate the isolation of lamina propria mononuclear cells from murine colonic tissue by enzymatic digestion using collagenase E and silica-based density separation media gradient separation. The isolated mononuclear cells can be used for subsequent downstream analysis.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Preparation of Solutions 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. Prepare Colon Buffer the day prior to…

Materials

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