Isolation of Murine Lymph Node Stromal Cells

Summary

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Abstract

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host.

Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Introduction

Lymph nodes are specialized compartments where adaptive immune responses against foreign and self-antigens are initiated and coordinated. The procedure presented here describes a short enzymatic digestion combined with automated mechanical pipetting to obtain lymph node single cell suspension and gain access to viable lymph node stromal cells that maintain the surface expression of several molecules.

Lymph node stromal cell form the scaffold of the lymph node and fulfill three major functions: first they filter body fluids to sample antigens, pathogens and their pathogen associated molecular pattern (PAMPs), as well as cytokines and danger ....

Protocol

In this video publication and protocol, all animal procedures were conducted in accordance to the animal protocol approved by the Cantonal Authority Basel-Stadt, Switzerland.

1. Lymph Nodes Preparation and Digestion

1. Pre-heat water in a beaker to 37 °C on a magnetic stirrer with heating plate.
2. Prepare Basic Medium as following: DMEM medium (without pyruvate) supplemented with 2% FCS, 1.2 mM CaCl₂ and Pen/Strep (100 units of penicillin, 100 µg of streptomycin).
3. Sterilize all dissection instruments before use.
4. Euthanize lymph node donor mice per CO₂ asphyxiation and asep....

Results

The present protocol is a modified digestion protocol published by Link et al., 2007⁶ with a shorter digestion time (45 min maximum) due to mechanical disaggregation with an automated multichannel pipette. In addition, the procedure is more standardized, minimizes degradation of surface markers on different lymph node stromal cells and allows the handling of more than one sample at the same time.

Collagenase IV and Collagenase D in Links protocol⁶ and current pro.......

Discussion

The study of lymph node stromal cells recently became a research focus due to the development of two published digestion protocols^6,13. Both protocols are adequate to gain single lymph node stromal cells but differ in the use of digestion enzymes and the time of digestion. Since stromal cells and their surface markers are sensitive to enzymatic digestion and mechanical stress, an optimized protocol is required.

The isolation of viable lymph node stromal cells from freshly dissected .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	LifeTechnologies-Gibco	41965-039
FCS			Final concentration 2%
CaCl2	Sigma	499609	Final concentration 1.2 mM
Collagenase IV	Worthington Biochemical Corporation		>160 units per mg dry weight, use at final concentration of 1 mg/ml
Collagenase D	Roche	11088882001	use at 3.5 mg/ml
DNAse I	Roche	11284932001	use at 40 µg/ml
Stiring magnets	FAUST		5 mm long-2 mm ø
Polystyrene round bottom tubes 5 ml	Falcon-BD Bioscience
Magnetic stirrer with heating function	IKA-RCT-standard	9720250
Petri dishes 100 mm, sterile	TPP	6223201
25 G needles	Terumo
anti mouse CD45 Ab	Biolegend		Clone 30-F11
anti mouse CD11c Ab	Biolegend		Clone N418
anti mouse Podoplanin Ab	Biolegend		Clone 8.1.1
anti mouse CD31 Ab	Biolegend		Clone MEC13.3
Eppendorf Xplorer plus, Multichannel	Eppendorf	4861 000.821/830	1.250 µl max. volume
anti mouse CD140a	Biolegend		Clone APA5
anti mouse CD80	Biolegend		Clone 16-10A1
anti mouse CD40	Biolegend		Clone 1C10
anti mouse I-Ab	Biolegend		Clone AF6-120.1
anti mouse CD274 (PD-L1)	Biolegend		Clone 10F.9G2
LIVE/DEAD Fixable Near-IR Dead Cell stain kit	Invitrogen	L10119