Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

The overall goal of this procedure is to accurately measure the glycolytic rate of cells using extracellular flux analysis. This method can help resolve ambiguities in the field of extracellular flux analysis, specifically the use of total extracellular acidification to find glycolytic rate. The main advantage of this technique is it allows quantitative correction of the total extracellular acidification rate to find both respiratory and glycolytic proton production rates.

Two, to measure the buffering power with an extracellular flux instrument, first distribute 50 microliters of diluted acid into each of the four A, B, C, and D ports of the measurement probe cartridge. Next program, the extracellular flux instrument to run an extracellular flux assay with a standard calibration cycle followed by two cycles for each of the four port additions. Then load the prepared cartridge into the machine and calibrate the instrument according to the software instructions.

When prompted by the program, remove the ca containing plate and insert the plate containing the assay medium. Then continue the program using the average of eight to 10 data points obtained at a steady state from before and after each port edition. To calculate the cumulative difference in the pH caused by each injection of standard acid.

Plot the change in pH against the M of protons contained in the seven microliter volume trapped by the measurement probe. The linear slope is the buffering power in milli pH units. Parols protons alternatively load ports A, B, and C with assay medium, followed by addition of the appropriate concentration of hydrochloric acid into Port D as indicated in the table.

Then measure the pH using the four technical replicates to generate each point of a five point standard curve in the 20 experimental wells of the extracellular flux assay plate. To measure the buffering power with an external pH meter warm the pH standards assay medium and 0.1 molar hydrochloric acid to 37 degrees Celsius in a water bath, warm the pH probe in calibration buffer to 37 degrees Celsius while the other materials are warming. Calibrate the probe at 37 degrees Celsius according to the manufacturer's instructions and aliquot 10 milliliters of the assay medium into a small container.

Immerse the pH probe in the medium for continuous monitoring. Then add 0.1 molar hydrochloric acid to the assay medium to 10 to 20 microliter aliquots at a time, stirring the medium. After each edition, allow the pH to stabilize for a few seconds after each edition of acid and record the pH measurement as demonstrated in the table.

Perform a sufficient number of additions to ensure an accurate slope calculation and to cover the pH range expected during the experiment 30 minutes prior to the assay. Wash the appropriate adhere cell population three times by gently aspirating the medium, followed by the slow addition of 500 microliters of KRPH to each well after the third wash. Incubate the cells at 37 degrees Celsius under air for 30 minutes to start the assay.

Replace the medium in each well with 500 microliters of fresh KRPH containing 500 units per milliliter of carbonic anhydrase with or without glucose pipette. 50 microliter aliquots of each experimental compound into the cartridge ports of an extracellular flux sensor cartridge at the appropriate concentrations and run a standard extracellular flux assay for determining the respiratory control ratio in this figure. The data for a typical experiment are shown using the last 10 measurement points from the point to point recording of both the oxygen consumption rate and the pH for the calculations here.

The respiratory proton production and glycolytic proton production rates calculated using individual extracellular acidification and oxygen consumption rates to determine the native conditions prior to the port. A edition of oligo mycin are shown the native rates of respiratory and glycolytic acidification. The rates following oligo mycin edition and the rates following FCCP edition as calculated from the table are displayed in the graph clearly demonstrating how the proportions of respiratory and glycolytic acidification change in response to changes in the substrate.

For this procedure, it's important to remember to do the acid at the same temperature as the planned experiments, typically 37 degrees Celsius following this procedure. Other assays like lactate measurement can be used to validate the calculated glycolytic rate. After watching this video, you should have a good understanding of how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic extracellular acidification using either internal or external acid calibration.

Don't forget that working with mitochondrial poisons can be extremely hazardous and precautions such as wearing gloves and measuring out dry reagent in the chemical fume hood should always be taken when performing this procedure.