Oct 08, 2018
Glycolysis Stress Test for Monocyte Glycolytic Function
- 1University of Memphis
Protocol Citation: Brandt D Pence 2018. Glycolysis Stress Test for Monocyte Glycolytic Function. protocols.io https://dx.doi.org/10.17504/protocols.io.ufaetie
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 08, 2018
Last Modified: October 08, 2018
Protocol Integer ID: 16578
Materials
MATERIALS
Cell-TakCorningCatalog #354240
Seahorse XFp FluxPakAgilent TechnologiesCatalog #103022-100
Seahorse Base Medium DMEMAgilent TechnologiesCatalog #102353-100
200 mM L-GlutamineMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7513
0.1 M Sodium Hydroxide SolutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #71395
Seahorse XFp Glycolysis Stress TestAgilent TechnologiesCatalog #103017-100
0.1M sodium bicarb buffer pH 8.0 sterile filtered
Pipettes and P1000 P200 P10 tips
Day Prior to Assay
Day Prior to Assay
Turn on XFp analyzer to warm up overnight
Hydrate XFp sensor cartridge
Add 200 μl XF calibration solution (included with FluxPak) to each well.
Add 400 μl sterile PBS or H2O to each moat.
Incubate overnight at 37oC in non-CO2 incubator.
Materials to Prepare
Materials to Prepare
Seahorse DMEM Media
Add 50 μl pyruvate, 50 μl L-glutamine, to 5 ml Seahorse Base Medium. Sterile filter before use.
Cell-Tak coated XFp plate
Prepare Cell-Tak. Add Cell-Tak and sodium hydroxide to 0.1 M sodium bicarbonate so that each well will receive 0.56 μg Cell-Tak, and sodium hydroxide concentration will be 0.63 mM. Cell-Tak concentration varies by batch, so calculations for each new batch will need to be performed. Add 25 μl Cell-Tak solution to each well. Plate can be stored at room temperature (at least 20 minutes) until use (for a few hours). Plates can also be prepped prior to assay day by incubating plates in Cell-Tak (at least 20 minutes), then aspirating remaining solution, air drying, and storing at 4oC until use.
Monocytes
Monocytes should be prepared as directed in the monocyte isolation protocol. Isolated monocytes should be diluted to a concentration of 3×106 cells / ml in prepared Seahorse DMEM media prior to use in the assay. Use monocytes immediately.
Procedure
Procedure
Aspirate Cell-Tak solution if not already done.
Add 50 μl medium to wells A and H and 50 μl cells to wells B-G. Samples are generally run in duplicate or triplicate on each plate. Cell number is 1.5×10^5 cells per well.
Place plate in the carrier and place in centrifuge. Spin 300×g for 1 minute without brake.
Add 130 μl assay medium to each well A-H (final volume 180 μl).
Incubate plate at 37oC in non-CO2 incubator for 1 hour.
While plate is incubating, perform steps 9-13
Prepare preliminary drug dilutions (mix by pipetting up and down)
100 mM glucose (blue cap) - add 300 μl medium
50 μM oligomycin (light blue cap) - add 288 μl medium
500 mM 2-deoxyglucose (green cap) - add 300 μl medium - vortex 1 minute
Prepare final drug dilutions
100 mM glucose - use as-is
10 μM oligomycin - 120 μl of 50 μM oligomycin in 480 μl medium
500 mM 2-deoxyglucose - use as-is
Remove sensor cartridge from incubator and remove and reinsert sensors briefly to clear air bubbles.
Fill cartridge:
Port A (all wells): 20 μl glucose (10 mM final concentration)
Port B (all wells): 22 μl oligomycin (1 μM final concentration)
Port C (all wells): 25 μl oligomycin (2 μM final concentration)
Port D (all wells): 27 μl 2-deoxyglucose (50 mM final concentration)
Note
We do two separate injections of oligomycin, although one seems to work (although less consistently). If one injection is desired, prepare oligomycin so that final concentration after the first injection is 2 μM.
Select Glycolysis Stress Test program on Seahorse XFp and calibrate sensor cartridge (remove lid).
Note
If two oligomycin injections are used, a custom program will need to be run.
After 1 hour cell incubation, remove utility plate from XFp and insert cell plate (remove lid).
Run Assay. 3 measurements per injection (including basal) is sufficient.
After run is completed, image each well by photomicroscopy or collect and isolate protein from each well to normalize cell numbers.
Data Analysis
Data Analysis
Data can be analyzed in the following manner. Averages or min/max for each condition can be used.
Glycolysis: (glucose ECAR) - (2-deoxyglucose ECAR)
Glycolytic Capacity: (oligomycin ECAR) - (2-deoxyglucose ECAR)
Glycolytic Reserve: (oligomycin ECAR) - (glucose ECAR)
Calculations are depicted in Figure 1.
Figure 1. Calculations for Glycolysis Stress Test.Notes