May 24, 2025
CalceinAM labeling of Extracellular Vesicles
- Sierra Palumbos1,
- Erika Holzbaur1
- 1University of Pennsylvania
Protocol Citation: Sierra Palumbos, Erika Holzbaur 2025. CalceinAM labeling of Extracellular Vesicles. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2pjd3g1y/v1
Manuscript citation:
https://doi.org/10.1101/2024.11.07.621551
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 23, 2025
Last Modified: May 24, 2025
Protocol Integer ID: 218843
Keywords: Extracellular Vesicle, EV, CalceinAM, calceinam labeling of extracellular vesicles protocol, extracellular vesicles protocol, intact vesicle, calceinam labeling, primary murine neuron, neuron
Funders Acknowledgements:
National Institute of Neurological Disorders and Stroke
Grant ID: R01-NS060698
National Institute of Neurological Disorders and Stroke
Grant ID: F32-NS129586
Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: MJFF-021130
Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: MJFF-15100
Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: MJFF-019411
Abstract
Protocol used to visualize intact vesicles isolated from primary murine neurons. Protocol used in Palumbos et al., 2025 and adapted from https://doi.org/10.1371/journal.pone.0317689
Materials
eBioscience‱ Calcein AM Viability Dye (ThermoFisher 65-0853-39)
PTFE Printed Slides (Cat #63430-04)
ExoEasy isolation kit (Qiagen Cat # 76064)
Troubleshooting
Isolate Extracellular Vesicles
Follow desired protocol for isolation of extracellular vesicles from conditioned media (e.g., differential ultraceltrifugation or ExoEasy kit).
Note
-A small volume of conditioned media (e.g., 1mL for ExoEasy) can be used and you will likely need to dilute to have best detection parameters
-The best isolation strategy will vary by cell type and vesicle population
Label Extracellular Vesicles
Filter 1X PBS through .2 µm filter
Note
Filtered PBS should be made fresh each time
Create series of dilutions of isolated EVs in filtered 1X PBS
Note
e.g., D1000 (diluted 1:1000), D10k, D100k, D1M
Place 10 µL of each diluted EVs in one well of PTFE printed microscope slides (Cat #63430-04 Electron Microscopy Sciences)
Note
Individual wells contain a bioadhesive surface which facilitates direct binding to slide.
Be sure to label which well has the corresponding dilution
Incubate diluted EVs in well for 00:05:00 at Room temperature
Wash each well 3X with 10µL filtered 1X PBS
Add 10 µL of 10µM Calcein AM (C1420) to each well (make working stock fresh each time)
Room temperature Incubate for 00:10:00 in the dark
Wash each well 3X with 10µL filtered 1X PBS
Add coverslip and seal with nail polish
Image Labeled Extracellular Vesicles
Find the center of each well
Use perfect focus to identify plane with labeled EVs
Calibrate orbital TIRF setup so puncta illumination is even over field and signal is crisp
Capture 6 images per well
Navigate to next well and capture
Note
By having 4X dilutions, you should be able to determine which dilution factor is best for your experiment. I would only recommend using a single dilution factor for analyses moving forward once protocol has been established
Make masks of captured images
Open images in FIJI
Run Trainable Weka Segmentation
Train classifier
Class 1 should indicate Calcein AM signal
Class 2 should indicate background and non specific signal
Individual puncta should be marked by class 1 and circled with class 2
Toggle overlay to determine if weka training is sufficient
Repeat training until overlay matches desired segmentation
Create result
*Do not select get probability, binary maps needed
Apply classifier to remaining images
Count Objects
Open binary weka output images in FIJI
Binarize
Object count
Can introduce size filter or circularity filter to be more stringent
Record count
Protocol references
Calderón-Peláez MA, Castellanos JE, Velandia-Romero ML. A protocol for loading Calcein-AM into extracellular vesicles from mammalian cells for clear visualization with a fluorescence microscope coupled to a deconvolution system. PLoS One. 2025 Jan 24;20(1):e0317689. doi: 10.1371/journal.pone.0317689. PMID: 39854328; PMCID: PMC11761115.