https://bridgeslab.sph.umich.edu/protocols/index.php?action=history&feed=atom&title=Measurement_of_Glycolysis_and_Respiration_Using_Seahorse 2026-06-01T21:12:26Z Revision history for this page on the wiki MediaWiki 1.45.1 https://bridgeslab.sph.umich.edu/protocols/index.php?title=Measurement_of_Glycolysis_and_Respiration_Using_Seahorse&diff=572&oldid=prev 2011-05-18T20:45:24Z

<p>wrote initial page</p> <p><b>New page</b></p><div>==Materials==<br /> *Non Buffered DMEM - Sigma cat#D5648 Dissolve one bottle (13.4g) in a litre of water. Adjust pH to 7-7.2 and filter into a sterile bottle.<br /> *FCCP - Prepare as 300 uM stock in DMSO. Need about 20 uL per plate<br /> *Oligomycin - Prepare as a 1 mM Stock in DMSO. Need about 20 uL per plate<br /> *XF24 plate<br /> <br /> <br /> ==Protocol==<br /> #Email Sydney Bridges to arrange a time to do the experiment. His email is sbridges@med.umich.edu<br /> #Trypsinise cells and resuspend in ~10 mL (for a 10 cm dish).<br /> #Count cells using hemocytometer or cell counter.<br /> #Adjust cell concentration to be 250 000 cells/mL (50K cells/200 uL) using normal growth media<br /> #Add 200 uL of cell suspension per well of a XF24 plate.<br /> #Let cells adhere for &gt; 30min and then add 800 uL of buffer<br /> #Incubate cells overnight.<br /> #Prior to experiment, aspirate 950 uL media then add 1 mL non-buffered DMEM.<br /> #Remove 1 mL media (leaving 50 uL)<br /> #Add 625 uL of non-buffered DMEM for a final volume of 675 uL.<br /> #Bring plate over to Brehm Room 6470 and place in non-CO2 incubator.</div>

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