Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier

Summary

We present a fluorescence assay to demonstrate that Lucifer Yellow (LY) is a robust marker to determine the apparent paracellular permeability of hCMEC/D3 cell monolayers, an in vitro model of the human blood-brain barrier. We used this assay to determine the kinetics of a confluent monolayer formation in cultured hCMEC/D3 cells.

Abstract

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of non-essential ions and toxic substances. Tight junctions (TJ) effectively seal the paracellular space in the monolayers resulting in an intact barrier. This study describes a LY-based fluorescence assay that can be used to determine its apparent permeability coefficient (P_app) and in turn can be used to determine the kinetics of the formation of confluent monolayers and the resulting tight junction barrier integrity in hCMEC/D3 monolayers. We further demonstrate an additional utility of this assay to determine TJ functional integrity in transfected cells. Our data from the LY P_app assay shows that the hCMEC/D3 cells seeded in a transwell setup effectively limit LY paracellular transport 7 days-post culture. As an additional utility of the presented assay, we also demonstrate that the DNA nanoparticle transfection does not alter LY paracellular transport in hCMEC/D3 monolayers.

Introduction

Blood-brain barrier (BBB) is the protective barrier limiting the influx of plasma components into the brain tissue and consists of brain endothelial cells along with supporting cells such as pericytes. The major role of BBB is to serve as a barrier that seals the space between peripheral blood and central nervous system (CNS) to maintain hemostasis of the neural microenvironment^1,2. The brain capillary endothelial cells effectively seal the paracellular pathway via formation of intercellular tight junctions (TJs)¹. This protective barrier allows glucose and selected nutrients to enter t....

Protocol

1.General hCMEC/D3 cell culture

1. Resuscitation of frozen cells
   NOTE: All cell culture maintenance and experiments were performed inside a sterile biosafety hood. Culture media, supplements and reagents were either purchased as sterile products or sterilized via filtration using a 0.22 µm membrane filter to prevent microbial contamination.
   1. Add 8.5 mL of collagen solution (0.15mg/mL) in a tissue culture flask (75 cm² growth area; referred henceforth as T75.......

Discussion

A key role of the BBB is to prevent the exchange of non-essential ions and toxic substances between the systemic circulation and the brain to maintain hemostasis of neural microenvironment. One of the characteristic features of the BBB is the ability of the capillary endothelial cells to form tight junctions (TJs) that effectively seal the paracellular route of transport. We demonstrated a LY P_app assay as a quantitative method to determine the apparent kinetics of TJ barrier formation in cultured hCMEC/D3 mon.......

Materials

Name	Company	Catalog Number	Comments
hCMEC/D3 cell line	Cedarlane Laboratories	102114.3C-P25	human cerebral microvascular endothelial cell line
gWizLuc	Aldevron	5000-5001	Plasmid DNA encoding luciferase gene
lucifer yellow CH dilithium salt	Invitrogen	155267
Transwell inserts with polyethylene terephthalate (PET) track-etched membranes	Falcon	353095
Tissue culture flask	Olympus Plastics	25-207
24-well Flat Bottom	Olympus Plastics	25-107
Black 96-Well Immuno Plates	Thermo Scientific	437111
S-MEM 1X	Gibco	1951695	Spinner-minimum essential medium (S-MEM)
EBM-2	Clonetics	CC-3156	Endothelial cell basal medium-2(EBM-2)
phosphate-buffered saline 1X	HyClone	SH3025601
Collagen Type I	Discovery Labware, Inc.	354236
Pierce BCA Protein Assay Kit	Thermo Scientific	23227
Cell Culture Lysis 5X Reagent	Promega	E1531
Beetle Luciferin, Potassium Salt	Promega	E1601
SpectraMax i3	Molecular Devices		Fluorescence Plate Reader
Trypan Blue Solution, 0.4%	Gibco	15250061
ZO-1 Polyclonal Antibody	ThermoFisher	61-7300
anti-GAPDH antibody	abcam	ab8245
Alexa Fluor680-conjugated AffiniPure Donkey Anti-Mouse LgG(H+L)	Jackson ImmunoResearch Inc	128817
12-well, Flat Bottom	Olympus Plastics	25-106
RIPA buffer (5X)	Alfa Aesar	J62524
Aprotinin	Fisher BioReagents	BP2503-10
Odyssey CLx imager	LI-COR Biosciences		for scanning western blot membranes