DMEM, low glucose, pyruvate, no glutamine, no phenol red

Question

Good afternoon 
 
 Can I use the DMEM with low glucose (1g/L, 4.5mM) and pyruvate, without no phenol red to measure the OxPhos (oligomycin, FCCP, Rotenone and Antimycin A) and Glycolysis (Glucose, oligomycin, and 2-DG) using the Cell Energy Phenotype Test (we will complete the kit with Rotenone, Antimycin A, Glucose and 2-DG)? That DMEM uses a sodium bicarbonate buffer system (3.7 g/L) and therefore requires a 5-10% CO 2 environment to maintain physiological pH. Is that an issue? We are planning to supplement it with Hepes to stabilize the pH. 
 We are planning on "starving" the cells for 45 min with Krebs before the glycolysis assay (With that DMEM medium) to deplete the glucose. Is that correct? 
 Best regards,

Courtney Watts · Accepted Answer

Hi Helene, 
 Thank you for contacting Cell Analysis Technical Support. Sodium bicarbonate cannot be used because it is a buffering agent with a very high pKa and, unless incubated in a CO2 incubator, will raise the pH of the assay medium to well above physiological levels. 
 We no longer carry the Cell Energy Phenotype Kit as it has been phased out. It has been replaced by the ATP Rate Assay , which can provide the same information and then some. However, from what you are describing, it sounds like you should run the Mito Stress Test and Glyco Stress Test assays. However, we also do have a newer assay called the Glycolytic Rate Assay , which is more physiologically relevant as there is no starvation period prior to running the assay. 
 If you can tell me a bit more about what you are looking to accomplish via your research with Seahorse assays, I may be able to point you in the right direction. 
 Thank you! Courtney 
 
 Courtney Nadeau Watts 
 Technical Support Scientist/Remote Engineer 
 Cell Analysis Products

DMEM, low glucose, pyruvate, no glutamine, no phenol red

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