May 22, 2025

Public workspaceApoptosis Detection with CellEvent Caspase-3/7

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzqd95vx1/v1
  • Sierra Palumbos1,
  • Erika Holzbaur1
  • 1University of Pennsylvania
Sierra Palumbos
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzqd95vx1/v1
Protocol Citation: Sierra Palumbos, Erika Holzbaur 2025. Apoptosis Detection with CellEvent Caspase-3/7. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzqd95vx1/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 21, 2025
Last Modified: May 22, 2025
Protocol Integer ID: 218698
Keywords: cell death, apoptosis, Caspase 3/7, CellEvent, CellPose, apoptosis detection with cellevent, apoptosis detection, cellevent caspase, cell death in primary cortical neuron, cellevent, cellpose machine, exosomal inhibition, following exosomal inhibition, cell, learning detection
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 describing methods used in Palumbos et al., 2025 to assay cell death in primary cortical neurons following exosomal inhibition. Approach combines CellEvent Caspase 3/7 reporter with Cellpose machine learning detection. Expanded methods applying these two approaches individually are available (see Invitrogen directions and Stinger and Pachitariu 2025).
Troubleshooting
Culture Neurons to DIV 7
On DIV0, plate 200k primary cortical neurons into 4 wells/condition of 12-well plate with glass-like bottom polymer (P12-1.5P, Cellvis).
Note
This can be done with any cell type. This protocol use culturing and dissection conditions as described in https://dx.doi.org/10.17504/protocols.io.81wgby723vpk/v1

Culture neurons with standard protocol until DIV7.
Prepare imaging media and warm in 37°C incubator with CO2.
Note
Imaging media- Hibernate E + B27 + Glucose
Need 4mL per well to be imaged.

Add Drugs to Plates
Prepare drug aliquots for concentration gradient to be added to 1mL maintenance media
Note
If no drug conditions are being used, skip steps 4-6

GW4869 (0µM, 1µM, 2.5µM, 5µM final concentration in 1mL maintenance media).
Add drug aliquots to corresponding well(s). Be sure to properly label
Return to 37°C incubator for 2 hours.
Prepare for Imaging
Prepare imaging media + CellEvent‱ Caspase-3/7. For 4mL, add 1 drop of CellEvent‱ Caspase-3/7 (R37111)
Warm media at least 30min in 37C waterbath
Wash plates 1X with prewarmed imaging media (no dye added)
Add 1mL of imaging media +CellEvent‱ Caspase-3/7 per well.
Pipetting
Add 1 µL of Hoechst per plate.
Pipetting
Immediately image, capturing DIC, 405, 488. Capture 10 fields of view per condition. Fields of view should be selected throughout the plate, without biasing based on caspase signal.
Note
This is experiment was conducted on a Leica DMI6000B inverted epifluorescence microscope equipped with a filter wheel for high-speed imaging and a climate-controlled chamber.

Imaging
Do not select field of view based on 488 signal, but rather using nuclear signal.
Capture 1 z-plane focusing on 405 signal. If you are also capturing DIC signal, this will likely require a separate z-plane.
Analysis Using Cell Pose
Create separate folders per channel (e.g., 405).
Open Cellpose3 (Stinger and Pachitariu 2025).
Note
Cellpose3 (Stinger and Pachitariu 2025) has thorough independent documentation that would be beneficial for other cell types and conditions.

Load folder containing images.
Check auto-adjust saturation.
Check- MASK ON, outlines on, single stroke.
Set nuclear size.
Segmentation- set diameter to 30 pixels.
Note
Other cell types or imaging conditions will have to adjust diameter size. Record all settings before moving forward with other images

Select model.
Other models- custom models- nuclei.
Run Cyto3.
Note
Before continuing with entire dataset, parameters should be adjusted based on signal, nuclear size, and confluency

Record ROIs counted.
Repeat steps 11-19 with CellEvent Caspase signal (488 channel folder)
All cell event ROIs should correspond with nuclear signal. To determine percent of nuclei that express CellEvent, take CellEvent ROI number divided by Hoescht ROI number.
Protocol references
Stringer, C., Pachitariu, M. Cellpose3: one-click image restoration for improved cellular segmentation. Nat Methods 22, 592–599 (2025). https://doi.org/10.1038/s41592-025-02595-5

CellEvent‱ Caspase-3/7 Detection Reagents