May 27, 2025

Public workspacepHrodo labeled synaptosome phagocytic assay

DOI
dx.doi.org/10.17504/protocols.io.dm6gpqnrdlzp/v1
  • Lucas Blasco-Agell1,2,
  • Meritxell Pons-Espinal1,2,
  • Jara Montero-Muñoz1,2,
  • Miquel Vila3
  • 1Department of Pathology and Experimental Therapeutics, Bellvitge University Hospital- IDIBELL, 08908 Hospitalet de Llobregat, Spain;
  • 2Institute of Biomedicine of the University of Barcelona (IBUB), Carrer Baldiri Reixac 15-21, Barcelona 08028, Spain;
  • 3VHIR-CIBERNED-ASAP
  • Vilalab Public
Miquel Vila
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DOI: dx.doi.org/10.17504/protocols.io.dm6gpqnrdlzp/v1
Protocol Citation: Lucas Blasco-Agell, Meritxell Pons-Espinal, Jara Montero-Muñoz, Miquel Vila 2025. pHrodo labeled synaptosome phagocytic assay. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpqnrdlzp/v1
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 27, 2025
Last Modified: May 27, 2025
Protocol Integer ID: 218937
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020505
Abstract
pHrodo labeled synaptosome phagocytic assay
Appropriate amount of human synaptosomes (SYNs) are thawed and centrifuged at full speed for 3-4 minutes at 4 ºC.
Pellet is resuspended and well-mixed in cold 100 mM of sodium bicarbonate, pH 8.5 to a concentration of 1 mg/mL.
SYNs are then incubated with amine-reactive pHrodo Red Succinimidyl Ester (SE) dye (Thermo Scientific) for 60 minutes at RT in a twist shaker (Labnet International).
Conjugated SYNs are rinsed with DPBS and centrifuged at full speed for 7 times. 10 μg/mL of SYNs were added to hMG plated on a CellCarrier‱-96 Ultra Microplate (Perkin Elmer‱)
Imaged using 20x objective of a Zeiss SP05 confocal microscopy, maintaining constant levels of temperature (37ºC) and CO2 (5%).
Four to five images are taken every minute for 5 hours using the phasecontrast and red fluorescence mode.
6 hours LPS pre-stimulated cells are employed as positive phagocytic controls.
Phagocytic Index (PI) is quantified using ImageJ, extracting background from the red fluorescent pHrodo.
Total fluorescence per image is divided by the total number of cells per image.
PI values for every hour of the time lapse are plot and the area under the curve (AUC) is quantified to perform statistical analysis.