Oct 27, 2022
Glycolysis stress test in organoids
- 1Icahn School of Medicine
Protocol Citation: gustavo.parfitt Parfitt 2022. Glycolysis stress test in organoids. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr46j3gmk/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: October 27, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 71934
Keywords: ASAPCRN
Abstract
Glycolysis stress test in organoids
Protocol materials
Seahorse XFe96 Spheroid Microplates Agilent TechnologiesCatalog ##102905-100
Step 1
Agilent Seahorse XF Base Medium Agilent TechnologiesCatalog #103334-100
Step 3
Seahorse XFp Glycolysis Stress TestAgilent TechnologiesCatalog #103017-100
Step 5
Seahorse XFe96 Spheroid Microplates Agilent TechnologiesCatalog ##102905-100 were coated with Laminin solution in L15+Bicarbonate: final concentration of 10 μg/mL .
Day 35 midbrain organoids were placed in the plates and media was changed every other day.
On the day of the assay Agilent Seahorse XF Base Medium Agilent TechnologiesCatalog #103334-100 supplemented with B27 + N2 was warmed at 37 °C and the pH as adjusted to 7.4. And 175 ul was added to the organoid plates.
The plate containing the organoids were warmed at 37 °C in a non-CO2 incubator for 30 min
Using the Seahorse XFp Glycolysis Stress TestAgilent TechnologiesCatalog #103017-100 kit the cartridges were loaded with loaded with Glucose (10mM), Oligomicyn (1uM) and 2-DG (50mM).
The run was done in
Equipment
Seahorse XFe96 Analyzer
NAME
Agilent
BRAND
BLE2100298
SKU
LINK
After the run the organoids were lysed in RIPA buffer and total protein was quantified using BCA kit.
The total protein was used to normalize the ECAR and OCR.