Mar 19, 2026
Protocol for Live-Cell Imaging of Astrocytes Exposed to HiLyte™ 488-labeled Human Recombinant α-Synuclein
- Elena Coccia1
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Elena Coccia 2026. Protocol for Live-Cell Imaging of Astrocytes Exposed to HiLyte™ 488-labeled Human Recombinant α-Synuclein. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge1jowv47/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: February 12, 2026
Last Modified: March 19, 2026
Protocol Integer ID: 243130
Keywords: astrocytes, live-cell imaging, α-synuclein, HiLyte™ 488, Hoechst 33342, synuclein on astrocyte, synuclein this protocol, cell imaging of astrocyte, synuclein, astrocyte, cell imaging, cell imaging at various time
Abstract
This protocol aims to investigate the effects of HiLyte™ 488-labeled human recombinant α-synuclein on astrocytes by performing live-cell imaging at various time points post-exposure.
Materials
96-well plate (Tissue culture treated)
Gelatin (0.1% solution) - 10 mL
Astrocyte media (AM media) - 200 mL
Mature astrocyte media (Specific formulation for astrocytes)
Reagents:
Protein HiLyte™ 488-labeled human recombinant α-synuclein - Fluorescently labeled protein for imaging
Stain Hoechst 33342 - DNA stain for cell nuclei visualization
Troubleshooting
Problem
Cells do not adhere to the plate
Solution
Ensure gelatin coating is applied correctly and not dried out.
Problem
Low fluorescence signal
Solution
Check the concentration of α-synuclein and Hoechst; ensure proper storage conditions.
Safety warnings
Handle all reagents and materials according to safety data sheets. Use personal protective equipment (PPE) including gloves and lab coats. Dispose of all waste according to institutional guidelines.
Cell Plating
Astrocytes are plated at a density of 10,000 cells per well in a 96-well plate coated with 0.1% gelatin.
Preparation of Gelatin Coating
1. Prepare a 0.1% gelatin solution by dissolving 0.1 g of gelatin in 100 mL of sterile PBS. 2. Add 100 µL of the gelatin solution to each well of the 96-well plate and incubate at room temperature for 1 hour. 3. Remove the gelatin solution and wash the wells with 200 µL of sterile PBS.
Plating Astrocytes
1. Collect astrocytes with Tryple and prepare a cell suspension of astrocytes and count cells 2. Add 100 µL of the cell suspension to each well of the gelatin-coated 96-well plate.
Media Change and Treatment
After 24 hours, change the media to mature astrocyte media and treat the cells.
Media Change
1. Aspirate the old media from each well and add 100 µL of mature astrocyte media.
Treatment with α-Synuclein and Hoechst
1. Prepare a working solution of HiLyte™ 488-labeled α-synuclein at a concentration of 2 µg/mL in mature astrocyte media. 2. Add 100 µL of the α-synuclein solution and 10 µL of Hoechst 33342 (10 µg/mL) to each well.
Live-Cell Imaging
Perform live-cell imaging at specified time points (3, 6, 12, and 24 hours post-treatment).
Imaging Procedure
1. At each time point, remove the media and wash the cells with 100 µL of pre-warmed PBS. 2. Add 100 µL of fresh mature astrocyte media to each well. 3. Place the plate in a fluorescence microscope and capture images.
Final Media Change
After the last imaging session (24 hours), change the media again.
1. Aspirate the media and wash with 100 µL of PBS. 2. Add 100 µL of fresh mature astrocyte media.