Aug 09, 2023

Public workspaceInducing proteostasis stress using G-TPP V.2

DOI
dx.doi.org/10.17504/protocols.io.dm6gp3jypvzp/v2
  • 1Lazarou Lab, WEHI
Louise Uoselis
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DOI: dx.doi.org/10.17504/protocols.io.dm6gp3jypvzp/v2
Protocol Citation: Louise Uoselis 2023. Inducing proteostasis stress using G-TPP. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3jypvzp/v2Version created by Louise Uoselis
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: August 09, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86200
Keywords: ASAPCRN
Abstract
Inducing proteostasis stress in HeLa cells with G-TPP to analyse proteostasis protection and repair.
Day 1
Day 1
Seed cells in standard growth media, aiming for a density of ~80-90% the following day.
Day 2
Day 2
Feed cells for Duration01:00:00 prior to starting treatment by aspirating the media from each well and replacing with a standard volume of growth media (eg. Feed with 2 mL of media if using a 6 well plate).

1h
Aspirate the media from each well/plate, and replace with standard growth media that has had G-TPP added at a final concentration of Concentration9 micromolar (µM) (for the DMSO control, add the same volume of DMSO as you added G-TPP to each well/plate).

Day 3
Day 3
Harvest the G-TPP treated samples and DMSO control samples after the desired incubation period (eg. 2 – 12 h).
For samples to be analysed followed after G-TPP removal, aspirate the treatment media from each sample, wash each sample 3x in an ample amount of room temperature PBS (eg. 3 mL per well for a 6 well plate).
After 3x PBS washes, add an appropriate volume of standard growth media to each well/plate (eg. 2 mL of standard growth media per well for a 6 well plate).
Day 4
Day 4
Harvest the 24 h recovery (24 h R) time point, at Duration24:00:00 after the washout step (steps 5 – 6) was performed.

1d
Replace the media on the plate for the 48 hour recovery (48 h R) samples with an appropriate volume of standard growth media (eg. 2 mL of standard growth media per well for a 6 well plate).
Day 5
Day 5
1d
1d
Harvest the 48 h R samples, Duration24:00:00 after the samples had fresh standard growth media added (step 8).

1d