Sep 13, 2022
Lysosome proteolysis analysis with DQ-BSA
- 1Icahn School of Medicine
- Ahfeldt Lab
Protocol Citation: gustavo.parfitt Parfitt 2022. Lysosome proteolysis analysis with DQ-BSA. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn2xmyg5d/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: September 13, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 69975
Keywords: ASAPCRN
Abstract
Protocol to analyze lysosomal proteolysis in astrocytes generated from iPSCs.
Protocol materials
TrypLE™ Select Enzyme (1X), no phenol redThermo FisherCatalog #12563029
Step 2
DQ™ Red BSA - Special PackagingThermo FisherCatalog #D12051
Step 7
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 TrypLE™ Select Enzyme (1X), no phenol redThermo FisherCatalog #12563029 for 00:05:00 at 25 °C
5m
300 x g, 25°C, 00:03:00
3m
Resuspend the pellet in 1 mL of astrocyte media (https://www.sciencellonline.com/astrocyte-medium.html).
Count cells and plate 10k cell per well in 100 µL of astrocyte media.
17h
Change the media to mature astrocyte media media in the next day.
2d
On the 3rd day, remove the media incubate with DQ™ Red BSA - Special PackagingThermo FisherCatalog #D12051 diluted in mature astrocyte media for 00:45:00
45m
Remove the media
Wash with PBS 3 times.
Add 100 µL of mature astrocyte media with vehicle or with Bafilomycin A1 100 nanomolar (nM)
Proceed to image in the Incucyte® SX5 Live-Cell Analysis System for 21:00:00 using the red channel
21h