Lysosome proteolysis analysis with DQ-BSA

Coat a 96 well plate Greiner 96 well Microplates Black with gelatin 0.1% or Geltrex LDEV Free hESC Quality 5 ml Thermo Fisher Scientific Catalog #A1413302 1:100

DIssociate extracted astrocytes with for 0h 5m 0s at 25°C

300x g,25°C

Resuspend the pellet in 1mL of astrocyte media (https://www.sciencellonline.com/astrocyte-medium.html)).

Count cells and plate 10k cell per well in 100µL of astrocyte media.

Change the media to mature astrocyte media media in the next day.

On the 3rd day, remove the media incubate with diluted in mature astrocyte media for 0h 45m 0s

Remove the media

Wash with PBS 3 times.

Add 100µL of mature astrocyte media with vehicle or with Bafilomycin A1 100nanomolar (nM)

Proceed to image in the Incucyte® SX5 Live-Cell Analysis System for 21h 0m 0s using the red channel