An Innovative Method of Endosome Isolation from Mouse Heart Tissues and Co-Culture with Cardiomyocytes

Summary

Endosomes are a collection of intracellular sorting organelles part of the endocytic membrane transport pathway. We established a new method for isolating endosomes from heart tissue using an automatic nanofiltration equipment and investigated the cellular uptake of endosomes by co-culturing them with primary cardiomyocytes.

Abstract

Endosomes are membrane-bound organelles playing essential roles in intracellular trafficking, signal transduction, and membrane recycling. Although their functions in cardiovascular biology are gaining recognition, efficient isolation of endosomes from cardiac tissue remains a significant technical challenge. We developed a rapid and high-yield protocol for isolating endosomes from mouse heart tissue by combining subcellular fractionation with an ultrafast nanofiltration isolation platform. This method includes sequential centrifugation, nanofiltration, and nanoporous membrane-based retrieval of vesicles. Isolated endosomes were characterized by western blotting, dynamic light scattering (DLS), and nano-flow cytometry (NanoFCM) analysis. To assess the bioactivity, purified endosomes were co-cultured with primary neonatal cardiomyocytes. The endosomal markers EEA1 and Rab7 were highly enriched in isolated vesicles. DLS analysis revealed a mean vesicle diameter of 187.30 ± 23.42 nm and a zeta potential of -26.60 ± 5.79 mV (n = 12). NanoFCM quantification yielded approximately 1.07 ± 0.31 × 10¹² particles/mL (n = 12) from 100 mg of heart tissue. Functional uptake of endosomes by cardiomyocytes was observed following in vitro co-culture, indicating preservation of vesicle integrity and activity. This integrated isolation platform provides a reproducible and efficient method for the extraction of high-purity endosomes from cardiac tissues. The protocol facilitates downstream biochemical and functional studies, offering a valuable tool for investigating the roles of endosomes in cardiovascular physiology and disease.

Introduction

Endosomes are essential intracellular organelles that regulate endocytosis, cargo sorting, and signaling pathways^1,2. Activated signaling receptors and other downregulated proteins are initially sorted into intralumenal vesicles (ILVs) within early endosomes (EEs), which then mature into multivesicular bodies (MVBs) or endosomal carrier vesicles (ECVs). These vesicles subsequently fuse with late endosomes (LEs), which serve as secondary sorting hubs for directing cargo toward lysosomal degradation or alternative pathways. ILVs may also be secreted as exosomes following endosome fusion with the plasma membrane<....

Protocol

All animal experiments were performed following the Guidelines of the National Institutes of Health on the Use of Laboratory Animals and with the approval of the Institutional Animal Care and Use Committee at the University of Houston.

NOTE: Prior to initiating the isolation procedure, it is essential to ensure that all required buffers have been freshly prepared and cooled to 4 °C. Sterilized instruments, tissue culture plates, and appropriate tubes should be arranged at designated workstations, with all materials maintained on ice or at 4 °C unless otherwise specified. Centrifuges and rotors must be pre-equilibrated to 4 °C....

Results

We examined the expression levels of markers for early endosomes, late endosomes, lysosomes, and autophagosomes among five groups: heart tissue lysate, nuclear fraction, mitochondrial fraction, other intracellular vesicles from step 2.6 and step 3.6, and endosomes from step 2.5 and step 3.6. Western blot result showed EEA1 (Early Endosome Antigen 1) had the highest expression level in endosomes and was almost unexpressed in other groups (Figure 2A,B). Rab7 was also expressed.......

Discussion

In this study, we present a rapid and reproducible method for isolating high-purity endosomes from mouse cardiac tissues by combining subcellular fractionation with an ultrafast isolation platform¹⁴. Traditional approaches for endosome isolation, such as density gradient centrifugation¹² or immune isolation, are often time and labor-intensive and require large quantities of tissue or specialized antibodies. By integrating a mechanical homogenization protocol with the nanofi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
µ-Slide 8 Well high	ibidi	80806-90
1.5 mL graduated microcentrifuge tube	USA Scientific	1615-5500
10x Tris/Glycine Buffer	Bio Rad	1610734
4x Laemmli Sample Buffer	Bio Rad	1610747
Benchtop centrifuge	Eppendorf	Eppendorf Centrifuge 5810R
Bovine serum albumin	Sigma	A9418
cell strainer	Corning	CLS431750	Pore size 40 μm, sterile
CELLSTAR Tissue Culture Dishes	VWR	82050-546	Polystyrene, Sterile, 60 mm ´ 15 mm
ChemiDoc imaging system	Bio Rad	ChemiDoc XRS+
Cleaning Solution	NanoFCM	17159
DMEM for Pierce Primary Cell Isolation Kits	Fisher Scientific	PI88287
DTT	Fisher Scientific	BP172
Dulbecco’s phosphate-buffered saline (PBS)	Thermo Scientific	10010023
ECL western blotting detection reagent	Bio Rad	1705061
EDTA	Sigma	E4884
EEA1	Cell signaling	3288S	WB (1:1000)
EGTA	Sigma	E-4378-25
EID	EXODUS	MA03 EID
ERp72	Cell signaling	5033S	WB (1:1000)
Ethanol	UH Research Store		200 Proof, 5 Gallon
EXODUS H-600	EXODUS	H-600
GAPDH	Proteintech	10494-1-AP	WB (1:5000)
Hank’s balanced salt solution (HBSS)	Thermo Scientific	14175095
HEPES	Sigma	H3375
Homogenizer	Fisher Scientific	15340167
KCl	Sigma	P3911
Lamp1	Abcam	ab320851	WB (1:2000)
LC3	Cell signaling	2775S	WB (1:1000)
Litesizer 500	Anton Paar	Litesizer 500	Other equipment such as Zetasizer (Malvern) or DLS  (Wyatt) can be used
mCherry	Biorbyt	orb66657	WB (1:500)
MgCl2	Fisher Scientific	BP214-500
NaCl	Sigma	S7653
NanoFCM	NanoFCM	Flow NanoAnalyzer
Nanospheres Size Standards	Thermo Scientific	3020A
Nikon AXR confocal microscope	Nikon	AXR
Paraformaldehyde solution 4% in PBS	Santa Cruz Biotechnology	sc-281692
Phalloidin Labeling Probes	Invitrogen	A22284	IF (1:500)
Pierce BCA Protein Assay Kits	Thermo Scientific	23227
Pierce Primary Cardiomyocyte Isolation Kit	Thermo Scientific	88028
Polyethersulfone syringe filter	Millipore	SLHPR33RS	Pore size 0.45 μm, sterile
Precast Protein Gels	Bio Rad	4561093
QC Beads	NanoFCM	QS2503
Rab7	Cell signaling	9367S	WB (1:1000)
RIPA Lysis and Extraction Buffer	Thermo Scientific	89900
Roche PhosSTOP (PI Cocktail)	Roche	4906837001
Silica Nanospheres	NanoFCM	S16M-Exo
Sucrose	Sigma	84097
Swinging-bucket rotor	Beckman Coulter	SW55Ti
Trans-Blot Turbo Transfer System RTA Transfer Kits	Bio Rad	1704270
Tween 20	Bio Rad	1662404
Ultracentrifuge	Beckman Coulter	Optima XE-90
Ultracentrifuge tubes	Beckman Coulter	326819
Ultrasonic Cleaning Bath	Branson Ultrasonics	CPX952238R
VWR Tube Cent 50 mL	VWR	89039-656
VWR Tube Centrifuge 15 mL	VWR	89039-666
Zeta Potential Reference Material	Anton Paar	175108