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^3,4 . In parallel, the autophagy pathway delivers cytosolic material to LEs via autophagosomes, which acquire degradative capacity upon fusion with late endocytic compartments⁵. Endosomes are now recognized as critical regulators of receptor recycling⁶, ion channel localization⁷, exosome biogenesis⁸, and intercellular communication⁹-processes that are especially pertinent in specialized cells such as neurons and cardiomyocytes.

Emerging evidence has highlighted the pivotal role of endosomal pathways in cardiovascular function. Eps15 Homology Domain protein 3 (EHD3) regulates endosomal recycling of key cardiac ion transporters, including the Na⁺/Ca²⁺ exchanger and L-type Ca²⁺ channels. EHD3 deficiency in mice leads to bradycardia, conduction anomalies, and altered Ca²⁺ handling¹⁰. Functional endosome-lysosome trafficking in vascular smooth muscle cells prevents phosphate-induced medial calcification, which is relevant to chronic kidney disease and atherosclerosis¹¹. Nevertheless, the functional roles of endosomes in cardiovascular diseases and their potential as therapeutic targets remain incompletely understood, warranting further investigation.

Multiple methods have enabled the biochemical isolation of functionally intact endosomes. Marsh and colleagues first combined density-gradient centrifugation with free-flow electrophoresis to recover early and late endosomes, preserving their acidification and structural integrity¹². Recent methodologies, such as the sucrose gradient approach, offer a standardized route for separating early and late endosomal populations, essential for downstream proteomic and lipidomic studies¹³. Spin-column-based commercial kits have been developed for high-throughput applications.

Exosome isolation via the automatic ultrafast-isolation system utilizes innovative ultrasonic nanofiltration technology, which has a dual-membrane nanofiltration system integrated with periodic negative pressure oscillation and double-coupled harmonic oscillations to enable the isolation of high-purity exosomes from biofluids¹⁴. Here, we combined subcellular fractionation with endosome isolation using the automatic ultrafast-isolation system to achieve a new method for rapid endosome isolation with high yield and high purity from mouse heart tissue.

Furthermore, we co-cultured the extracted endosomes with primary cardiomyocytes (CM) in vitro and found that the exogenous endosomes entered the CM successfully. Using an endosome reporter, in which the endosome-localized protein Numb was fused with mCherry red fluorescent protein^15,16, we observed mCherry fluorescence localized inside CMs with a puncta pattern.

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 for a minimum of 2-3 h before use. The fractionation buffer should be prepared before the day of the experiment and stored at 4 °C, and kept on ice during the experiment until needed. Unless indicated otherwise, all incubation and centrifugation steps should be conducted at 4 °C.

1. Heart lysate preparation

1. Disinfect the surgical area using 70% ethanol, followed by placement of a sterile absorbent pad on the work surface, upon which sterilized surgical instruments are arranged.
2. Euthanize mice (C57BL/6 males, 8-12 weeks, 20-25 g, and mCherry: NumbKI males, 8-12 weeks, 20-25 g) via CO₂ inhalation (10-30% CO₂ for 3-5 min) or by an alternative method approved by the relevant institutional animal care and use committee.
   CAUTION: Please operate in an open space and ensure the CO₂ cylinder closes in time to prevent leakage. Mice should be monitored during euthanasia to confirm loss of consciousness and proper procedure. Once unconscious, use a secondary method, such as cervical dislocation, thoracotomy, or exsanguination, to ensure death.
3. Position the mouse in a supine position and clean the thoracic area with 70% ethanol.
   1. Open the thoracic cavity rapidly to harvest the heart, and transfer the heart into ice-cold Hank's Balanced Salt Solution (HBSS) in a 60 mm dish placed on ice.
   2. Remove the residual blood gently from the cardiac chambers using tweezers and wash the heart very well until no more blood remains in the chambers. Keep the heart on ice throughout processing.
      NOTE: Save 50-100 mg of heart tissue for western blot (WB) analysis as a positive control. The heart can be snap-frozen in liquid nitrogen and stored at -80 °C for up to 6 months.
4. Add 8 mL of fractionation buffer (Table 1) to a 50 mL tube on ice. Transfer 100-120 mg of fresh or frozen mouse cardiac tissue into a tube containing the fractionation buffer.
   NOTE: Any part of the mouse heart tissue can be used to extract endosomes. A minimum of 100 mg of cardiac tissue is required to yield sufficient endosomes for subsequent analyses. To ensure consistency across the experimental groups, the tissue should be accurately weighed before the addition to the Fractionation Buffer.
   CAUTION: Wear appropriate PPE when operating the fractionation buffer to avoid any direct contact with the body.
5. Mince the tissue into about 1 mm³ pieces using microsurgery scissors in fractionation buffer on ice all the time.
6. Homogenize with an electric homogenizer at medium speed, in a 40 s work 30 s stop cycle. Homogenize for 5-7 cycles (Figure 1A).
   NOTE: A thorough homogenization process is essential and will help increase the final concentration of endosomes.
7. Sonicate the homogenized heart lysate for 10 min in the ice water bath with an ultrasonic bath (Figure 1A).

2. Subcellular fractionation

1. Place a 40 µm cell strainer over a 50 mL centrifugation tube and filter the homogenized heart lysate through the strainer (Figure 1A).
2. For the nuclear pellet: centrifuge the heart lysate at 1,000 × g for 5 min at 4 °C. Transfer the supernatant and place it in a fresh labeled tube.
   NOTE: Collect the pellet, which is the nuclear fraction, and resuspend the nuclear pellet in 200 µL WB Lysis Bufferif the nuclear fraction is needed for WB. Gently pipette the nuclear pellet with radio immunoprecipitation assay (RIPA) buffer up and down 20 times and sonicate for 10 min. Centrifuge the nuclear lysate at 14,000 × g for 10 min. Collect the supernatant and store it at -80 °C for further use. Step 2.2 is optional; it can be skipped if only vesicles are required.
3. For the mitochondrial fraction: centrifuge the supernatant again at 10,000 × g for 10 min at 4 °C. The pellet contains the mitochondrial fraction. Transfer the supernatant and place it in a fresh ultracentrifuged tube.
   NOTE: If a mitochondrial fraction is desired for WB, save the pellet as step 2.2 as described in the previous NOTE.
4. Carefully balance the tubes. Weigh each tube and add additional cold fractionation buffer to the lighter tube to ensure equal weight. Precool the ultracentrifuge rotor and adaptors for 2-3 h at 4 °C. Ultracentrifuge the supernatant at 160,000 × g for 30 min at 4 °C.
   NOTE: During the ultracentrifugation step, it is critical to ensure that all ultracentrifuge tubes are properly balanced and filled with buffer to approximately 0.5-1 cm below the rim. Inadequate filling may result in tube deformation or collapse due to the high centrifugal forces applied.
5. Remove the supernatant into a fresh tube. The supernatant contains endosomes. Set up a 10 mL syringe attached to a 0.45 µm syringe filter on top of a 50 mL centrifugation tube and filter the supernatant into the tube. Add 5 mL of sterilized PBS buffer to dilute the solution and avoid clogging the filter.
   NOTE: The filtering step ensures that all endosomes in the supernatant are <450 nm when starting the isolation steps.
6. Resuspend the pellet with 5 mL of sterilized PBS buffer from step 2.4 and pipette up and down at least 20 times to mix the pellet well. The pellet contains other intracellular vesicles. Set up a 10 mL syringe attached to a 0.45 µm syringe filter on top of a 50 mL centrifugation tube and filter the pellet solution into the tube. Add an additional 5 mL of sterilized PBS buffer to dilute the solution and avoid clogging the filter.
   NOTE: The filtering step ensures that all vesicles from the pellet are <450 nm when starting the isolation steps.

3. Isolation process of endosomes and other intracellular vesicles

1. Dilute the filtered supernatant from step 2.5 and the filtered pellet solution from step 2.6 with sterilized PBS buffer to a final volume of 30 mL in 50 mL tubes, respectively. Store the samples in 4 °C (Figure 1A).
   NOTE: The samples can be stored at 4 °C for 24 h. If there is not enough time to carry out the following steps on the same day, they can be stored in a refrigerator at 4 °C and then wait for the next day to continue the experiment.
2. Turn on the instrument and start the daily cleaning program, which usually takes 20 min.
3. Choose the Medium EXODUS isolation device (M EID) for isolation. Install a 50 mL tube adapter and put the 50 mL tube with the sample into the sample position. Tap EID out on the screen and load M EID.
   NOTE: The Medium EID is for a 20-50 mL sample, recommending 30 mL.
4. Set up program:
   1. Enter the sample name and sample volume.
   2. Select sample type: Plasma.
   3. Select program: Plasma. Strong-MA. A03.
   4. Select a centrifuge tube: 50 mL.
   5. Turn on High Purity Mode.
   6. Select Program Option 3000.
   7. Tap OK to start.
      NOTE: The entire isolation process takes 1-2 h to complete. A higher concentration of samples will take a longer time.
5. Recover endosomes and other intracellular vesicles:
   1. Retrieve the EID and bring it to the hood for the next steps.
   2. Transfer 200 µL of sterilized PBS buffer into the EID with a 200 µL pipette. Rinse both sides of the nanoporous membrane 20 times, respectively.
   3. Collect the endosomes or other intracellular vesicles, resuspend in 200 µL in a 1.5 mL tube. Store it at 4 °C if used in 24-48 h, or at -80 °C for long-term storage.
      NOTE: The eluate contains the endosomes or other intracellular vesicles and is used for further analysis and co-culture with cardiomyocytes. The sample of endosomes is stable when it is stored at -80 °C for 5 months, based on the data obtained.
6. Elute endosomes and other intracellular vesicles for WB:
   1. Retrieve the EID. Transfer 80 µL of WB lysis buffer into the EID. Rinse both sides of the nanoporous membrane 20 times, respectively.
   2. Incubate the EID on ice for 30 min to ensure complete solubilization, pipette buffer inside the EID every 10 min. Collect the endosomes or other intracellular vesicles lysate in 1.5 mL tubes. Centrifuge for 10 min at 14,000 × g at 4°C. Collect and store supernatant in -80 °C for long-term storage. Discard the pellet if any pellet remains in the tube.

4. Endosome protein quantification and vesicle characterization

1. Western blot analysis
   1. Save 5 µL from each sample to quantify protein concentration using a bicinchoninic acid (BCA) assay.
   2. Dilute samples in 4× Laemmli sample buffer and mix thoroughly by vortexing. Heat the samples at 95 °C for 10 min to denature proteins.
   3. Centrifuge the denatured samples at 14,000 × g for 10 min at 4 °C and collect the resulting supernatant for analysis.
   4. Assemble polyacrylamide gels in the electrophoresis apparatus and fill the chamber with sodium dodecyl sulfate (SDS) running buffer.
   5. Load a molecular weight marker and the prepared protein samples onto the gel, selecting an appropriate acrylamide percentage based on the molecular weight of the target proteins.
      NOTE: Typically, 20 µg of protein is loaded per well for both heart tissue lysates and endosome lysates.
   6. Run the gel at 6 V for approximately 120 min or until the dye front reaches the bottom of the gel.
   7. Transfer proteins from the gel to a polyvinylidene fluoride (PVDF) membrane using a western blotting transfer system.
      NOTE: The wet transfer method also works well.
   8. Rinse the PVDF membrane briefly with Tris-buffered saline containing 0.05% Tween-20 (TBST).
   9. Block the membrane with TBST supplemented with 1% bovine serum albumin (BSA) for 1 h at room temperature.
   10. Incubate the membrane overnight at 4 °C with primary antibody (Table of Materials) specific to the selected endosomal marker, using gentle rocking.
   11. The following day, wash the membrane three times with TBST, each wash lasting 10 min at room temperature on a rocker.
       NOTE: Proper washing is essential to reduce nonspecific background; ensure the membrane remains fully hydrated throughout the process.
   12. Incubate the membrane with the appropriate secondary antibody (Table of Materials) for 1 h at room temperature.
   13. Wash the membrane three times with TBST, each wash lasting 10 min on a rocker at room temperature.
   14. Apply chemiluminescent substrate to the membrane for the detection of antibody-bound proteins.
   15. Using a gel imaging system to expose the membrane and take images of the proteins.
2. Dynamic light scattering (DLS) analysis
   1. The particle size and zeta potential of endosomes are measured using the dynamic light scattering (DLS) technique¹⁷. using a Litesizer 500 (Anton Paar) equipped with Kalliope software version 2.10.4. DLS measurements are performed in triplicate using a 658 nm laser with a 90° detection angle at 25 °C.
   2. Make a dilution of "size standard nanospheres" in ultrapure water, add 1 mL volume in the disposable cuvette, and place it in the chamber.
      NOTE: Use standard nanospheres (either small or large) according to the expected size of the unknown sample.
   3. Open the DLS software and click on the new measurement tab. Select particle size from the measurement mode. Name the file in the Untitled field and put the input parameters (solvent, temperature, etc.), and click on start. After determining the size of the standard particles, take out the cuvette from the chamber.
   4. Make a dilution of "zeta potential reference material" in ultrapure water, place it in the zeta potential omega cuvette, and place it in the cuvette chamber. Click on the new measurement tab in the software. Select zeta potential from the measurement mode. Name the file in Untitled field and put the input parameters (solvent, temperature, etc.), and click on start.
   5. For the determination of particle size and zeta potential of endosomes, dilute the endosome sample in ultrapure water, put it in the respective cuvettes, and place them in the cuvette chamber. Take measurements in the same way as described for the standards.
      NOTE: Make a dilution of the unknown samples according to the concentration of the samples in ultrapure water.
3. NanoFCM analysis
   NOTE: Endosome particle concentrations are determined using NanoFCM Flow NanoAnalyzer with Professional Suite V2.3 software¹⁸. Particle signal acquisition is performed for 2 min using a 488 nm blue laser set to 20 mW with 0.2% SS decay and/or a 638 nm red laser set to 8 mW with 10% SS decay, at a sampling pressure of 1.0 kPa modulated and maintained by an air-based pressure module. The sample is focused at 1.4 µm using ultrapur water as the sheath fluid via a gravity feed. The optimal laser alignment is tested and calibrated using silica nanosphere cocktail QC beads (S16M-Exo). Size calibration measurements are performed prior to analysis using S16M-Exo size standard beads (silica nanoparticle cocktail containing 68, 91, 113, and 155 nm diameters of particles with known concentration 2.17 × 10¹⁰/mL).
   1. Concentration standard acquisition:
      1. Start up and initialize the fluidics of nanoFCM. Dilute the QC beads (1:100) in 0.22 µm membrane-filtered ultrapure water, add to a 0.5 mL tube, and place in the loading bay. Select QC FL SiNPs from Sample Inf dropdown menu and input the dilution factor of the QC beads sample.
      2. From the Sample Flow dropdown menu, select boosting to introduce the QC sample at a high speed and remove the cleaning solution from the system for 45 s. During boosting, set the laser power to 20/50 mV for the 488 nm blue laser and 20/100 mW for the 638 nm red laser, with 0.2% SS decay.
      3. Select sampling from the Sample Flow dropdown menu and do threshold adjustment for the large signal. During the sampling, adjust the signal condensing lens alignment manually and the laser focusing lens alignment from the software, if needed. Auto threshold adjustment for a large signal must remain between 50-120 for the side scatter channel and less than 75 for both fluorescence channels.
      4. Click Time to record to acquire the data for 120 s. Ensure that the region of interest (ROI) is between 1500-15000 when acquiring the data. Save the result file in the pop-up folder with the "nfa" extension and unload the sample tube.
         NOTE: After each sample run, use cleaning solution (1x) to remove the processed sample from the tubing by boosting it for 45 s. Then, dip the capillary tip in ultrpure water to remove residual cleaning solution from the outside of the capillary before loading the next sample.
   2. Size standard acquisition:
      1. Dilute the "size standard beads" (1:100) in 0.22 µm membrane-filtered ultrpaure water and place in the loading bay. Select 68-155 S16M-Exo from Sample Inf dropdown menu and input the dilution factor of the size standard beads sample.
      2. From the Sample Flow dropdown menu, select boosting to introduce the size standard beads and remove the cleaning solution from the system for a duration of 45 s. While boosting, set the laser power to 8/50 mV for the 488 nm blue laser and 20/100 mW for the 638 nm red laser, with 10% SS decay.
      3. Select Sampling from the Sample Flow dropdown menu and do threshold adjustment for a small signal. Four distinct peaks (68 nm, 91 nm, 113 nm, 155 nm) should be visible in the window. Click Time to record to acquire the data for 120 s. Save the result file in the pop-up folder with "nfa" extension and unload the sample tube.
   3. Blank standard acquisition:
      1. Use ultrapure water or (buffer/diluent of the sample) to take the measurement in the same process as described above, save the file with the name "blank" in the folder with "nfa" extension, and unload the sample tube.
   4. Determining particle concentration of endosomes
      1. Dilute the endosome sample in ultrapure water and place it in the loading bay.
      2. After boosting and sampling the sample, click Time to Record to start the measurements. If the threshold is not within the range of 50-120 for the side scatter channel, or the ROI is more than 15,000, then consider diluting the sample.
   5. Data analysis:
      1. Switch from the Acquisition tab to the Analysis tab in the software and open the saved "nfa" files. Set the concentration standard by opening the saved file of QC results date__QC FL SnNPs.nfa, set the threshold on large signal by clicking the set threshold tool.
      2. Select the particle concentration using the circle gating tool. Click Count STD and check the concentration of QC beads (2.17 × 10¹⁰/mL) before closing the window.
      3. To set the size standard, open the date_68-155 S16M-Exo.nfa file. Click the Size tool to open the standard curve generation window and set the threshold for the small signal by clicking on the set threshold tool.
      4. Select S16 exo 68-155 nm from the User Defined dropdown menu. Click Find Peaks to identify the peak SS intensities of the four-sized populations.
      5. Select the blank file and click on Set Blank to identify the number of false positives for removal of the sample. To analyze the endosome concentration, first select the sample and set the threshold on a small signal by clicking on the set threshold tool. Present the quantified data as a size (nm) and concentration (particles/mL) report.

5. Primary cardiomyocyte culture

NOTE: Isolate cardiomyocytes using a primary cardiomyocyte isolation kit, which provides sufficient reagents for the isolation of cardiomyocytes from up to 50 neonatal mouse or rat hearts. The kit also includes components that are suitable for supporting primary cardiomyocyte culture. All media and buffers should be removed with caution to avoid disturbing cells. Manual pipetting using a 1000 µL pipette tip is recommended, vacuum aspiration should be avoided. Hank's Balanced Salt Solution (HBSS) should be pre-chilled to 4 °C before initiating the procedure.

1. Reconstitute Cardiomyocyte Isolation Enzyme 1 (containing papain), available in the kit, by adding 2. mL of chilled HBSS to one vial. Mix gently for approximately 5 min or until the enzyme is fully dissolved. Keep the enzyme solution on ice throughout the procedure.
   NOTE: A single vial of reconstituted Enzyme 1 is sufficient for the isolation of cardiomyocytes from 10 neonatal hearts. It can be stored at -20 °C for up to 6 months and is stable for two freeze-thaw cycles. If stored at 4 °C, the solution should be used within 1 week to ensure enzymatic activity.
2. Place freshly dissected neonatal hearts into individual sterile 1. mL microcentrifuge tubes and immediately add 500 µL of ice-cold HBSS to each tube.
   NOTE: It is recommended to process one heart per tube to maximize yield and reproducibility.
3. Mince heart tissue into 1- mm³ fragments using sterile dissection scissors. Wash each sample twice with 500 µL of ice-cold HBSS to remove residual blood.
4. To each tube, add 200 µL of reconstituted Cardiomyocyte Isolation Enzyme 1 (papain) and 10 µL of Cardiomyocyte Isolation Enzyme 2 (containing thermolysin), available in the kit. Gently mix and incubate the tubes at 37 °C for 30-35 min.
   NOTE: Cardiomyocyte Isolation Enzyme 2 is provided as a suspension in HBSS and should be thawed on ice prior to use. Homogenize the enzyme by pipetting up and down to ensure a uniform suspension before being added to the samples.
5. Following enzymatic digestion, carefully aspirate the enzyme solution and wash each sample twice with 500 µL of ice-cold HBSS.
6. Add mL of Complete DMEM for Primary Cell Isolation to each tube. Gently triturate the tissue by pipetting up and down 25-30 times using a sterile 1.0 mL pipette tip. Minimize the formation of air bubbles during this step.
   NOTE: Disrupting tissue by pipetting improves cell yield. However, excessive pipetting may lead to cell damage.
7. Centrifuge the supernatant at 300 × g for 5 min.
8. Remove the Complete DMEM carefully with a pipette. Add 1 mL fresh Complete DMEM to the tube and pipette up and down 25-30 times using a sterile 1.0 mL pipette tip. Avoid air bubbles when pipetting.
9. After the tissue is primarily a single-cell suspension, add 0.5 mL of Complete DMEM for primary cell isolation to each tube to bring the total volume to 1.5 mL. Combine individual cell suspensions for the determination of cell concentration and cell viability.

6. Co-culture endosome with cardiomyocytes

1. Seed the cardiomyocytes in a 60 mm dish and add Complete DMEM to the total volume.
   NOTE: Each neonatal mouse heart is expected to be seeded in one dish, and the total volume of each well is expected to be 5 mL (Figure 1B).
2. Incubate the 60 mm dish at 37 °C in a 5% CO₂ incubator for 24 h. After 24 h, replace the medium with an equivalent volume of fresh Complete DMEM for Primary Cell Isolation containing Cardiomyocyte Growth Supplement diluted 1000x.
3. Transfer the endosome eluate produced by step 3 from -80 °C to -20 °C for 1 h. Then, put the endosomes on ice and thaw them gradually.
   NOTE: Gradual thawing on ice can make the membrane of endosomes more intact and reduce degradation.
4. After 48 h, mix every 100 µL of endosome eluate with 5 mL of fresh Complete DMEM and replace the old medium in each dish. Co-culture endosomes with cardiomyocytes for 24 h.
5. Wash the cardiomyocytes with 2 mL of cold sterile PBS. Add ice-cold lysis buffer to each dish with 80-100 µL lysis buffer. Scrape the cells using a cold plastic cell scraper and collect the cells in 1.5 mL tubes.
6. Perform the WB protocol described in step 4.1.
7. For immunostaining (IF) follow the steps described below.
   1. Seed cardiomyocytes in an 8-well chamber slide. Dilute the single-cell suspension with completed DMEM from 1.5 mL to 3 mL.
   2. Seed 300 µL of the suspension to each chamber. Repeat steps 6.2-6.4.
   3. Fix the cardiomyocytes with 200 µL of 4% paraformaldehyde (PFA) for 20 min at room temperature and wash with 200 µL of sterile PBS three times. The 8-well chamber slide is ready for IF.
      CAUTION: When handling 4% PFA, please wear appropriate PPE. Please discard 4% PFA in a proper container after use.

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