Murine Dermal Lymphatic Endothelial Cell Isolation

Summary

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

Abstract

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.

Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Introduction

The lymphatic system plays a pivotal role in human physiology. It is considered a vital homeostatic factor, that facilitates important functions, such as the maintenance of tissue-plasma fluid balance, immune surveillance, and lipid absorption¹, as well as recently-identified functions, such as the repair ability of the heart² and regenerative capacity of bone and hematopoiesis³. Despite the significant role of the lymphatic system, several aspects of the role of the lymphatics, as well as the molecular mechanisms governing certain physiological parameters and responses remain elusive. Moreover, l....

Protocol

Animal studies were performed according to the approved protocols by the Institutional Animal Care and Use Committee (IACUC) of Texas Tech University Health Sciences Center (TTUHSC) for the experiments at TTUHSC and by the Veterinary Administration of the Prefecture of Western Greece according to Directive 2010/63 for the experiments at the University of Patras. Diligently follow waste disposal regulations when disposing of animal waste materials.

1. Isolation of mouse dermis

1. Day 1
   NOTE: On Day 1; get the following materials and equipment ready: anesthesia chamber, animal hair clipper, isoflurane, fine fo....

Results

The method was developed to identify the protein expression of a small GTPase, RhoA, the coding gene of which was floxed in both alleles and deleted under the effect of the tamoxifen-inducible lymphatic endothelial-specific promoter Prox1-CreER^{T2 18}. The isolated LECs can be cultured for up to four generations, after which they become senescent. They have been frozen, thawed, and successfully stimulated with angiopoietin-2. Due to the limited number of cell passages, we have no.......

Discussion

The lymphatic system is an important regulator of the homeostatic function of the body, with the most important functions being the maintenance of fluid plasma, removal of cellular metabolism byproducts and toxic molecules, lipid absorption, and immune cell trafficking^1,19. The identification of appropriate markers has provoked a burst in new data in the lymphatics field, revealing novel functions of the lymphatic vasculature, such as their role in the repair and.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 μm Syringe Filters	Fisher Scientific	09-719C
100 mm Tissue Culture Dishes	Fisher Scientific	FB012924
60 mm Tissue Culture Dishes	Fisher Scientific	FB012921
Animal Hair Clipper	Wahl
Antibiotic-Antimycotic Solution 100x	Fisher Scientific	15240-062
Blunt Forceps	Fine Science Tools	11992-20
Bovine Serum Albumin	Sigma-Aldrich	A4919
Cell Strainer 40 μm	Fisher Scientific	542040
Cell Strainer 70 μm	Fisher Scientific	542070
CO2 Incubator
Collagen I, High Concentration Rat Tail	Corning	354249	dilute in 0.02 M Acetic Acid in H2O
Collagenase Type II	Life Technologies	17101-015
Dispase	Life Technologies	17105-041
DMEM	Fisher Scientific	11-995-073
DNAse I Solution (2,500 U/mL)	Thermo Scientific	90083
Dynal MPC-L Magnetic Particle Concentrator	Invitrogen	120-21D
EDTA	Sigma-Aldrich	3690
Endothelial Cell Growth Base Medium & Supplement (LEC medium)	R&D Systems	CCM027
Euthanasia chamber	Euthanex Corporation
Fine Forceps	Fine Science Tools	11255-20
Fine Scissors-Sharp	Fine Science Tools	14060-10
FITC anti-mouse F4/80 Antibody	Biolegend	123107
Goat Anti-Rabbit IgG Magnetic Beads	New England Biolabs	S1432S
LARC-A E-Z Anesthesia Induction Chamber	Euthanex Corporation
MagnaBind Goat Anti-Rabbit IgG Beads	Thermo Scientific	21356
Paraformaldehyde Solution 4% in PBS	Fisher Scientific	AAJ19943K2
Phosphate Buffered Saline (PBS)	Fisher Scientific	SH30256FS
Rabbit Anti-Mouse LYVE-1	ReliaTech GmbH	103-PA50
Rotating/Shaking Incubator
Round-Bottom Polypropylene Tubes	Corning	352063
Syringe Filters w 0.2 μm Pores	Fisher Scientific	09-719C
Trypsin-EDTA	Fisher Scientific	25-300-120