Making Medium and Setting Up Experiment

Today I learned more about the Seahorse XF Cell Mito Stress Test I will be conducting in the upcoming weeks. We are conducting this experiment in order to determine the mitochondrial function by directly measuring the oxygen consumption rate of the cell. Various compounds get injected to measure specific parameters using the modulators. The first step to this experiment was to make the medium.

On Saturday, the cells were plated and when I looked at them underneath the microscope today they appeared to be sparse due to their underestimated growth. On the plate, three sensors formed a triangle shape. This area surrounding the sensors have to have to most cells because that is the region the sensors detect.

Then, I aliquoted 10mL of medium into a 50mL tube so I could then find its pH and ensure the pH of the medium was at 7.4 and appropriate for the following tasks. The pH is important because from that we can add the glutamine and the pyruvate since the medium does not have those.

However, we do not have to add the glucose, because the medium already included that. It was important that we use a fresh batch of the glutamine because glutamine degrades faster than the pyruvate. Next, since the pH was too acidic, we had to add small amounts of 1mM NaOH in order to make the medium more basic. Since the NaOH has no sodium bicarbonate or hepes, we began to add 3mM of the NaOH at a time until we reached the desired pH. In addition to measuring the pH, the color of the medium also acts as in indication for determining the appropriate pH. Next time I will continue the prep before running the test.