The overall goal of this procedure is to accurately measure glycolysis and mitochondrial respiration in lymphocytes using an extracellular flux assay. This method can help answer key questions in the immunometabolism field, such as what metabolic changes occur in immune cells upon activation, differentiation, or during disease. The main advantage of this technique is that it allows for the efficient and real time analysis of metabolic profiles of over 90 samples at a time.
Though this method can provide insight into lymphocyte metabolism it can also be applied to other cells including non adherent cells that require plate addition with minimal disruption to their functionality. Generally, individuals new to this method will struggle because lymphocytes are fragile, and require gentle handling to maintain their functionality and viability. The day before the experiment lift the sensor cartridge of the extracellular flux analyzer and fill each well of the utility plate with 200 microliters of calibrant solution.
Next, lower the cartridge onto the plate, submerging the sensors in the calibrant solution and incubate the cartridge in a 37 degree Celsius incubator without carbon dioxide or nitrogen overnight. The next morning coat each well of a 96 well assay plate with 25 microliters of adhesive solution and incubate the plate at room temperature for 20 minutes. At the end of the incubation aspirate the adhesive and wash each well two times with 200 microliters of sterile water.
While the plate is air drying place a 70 micron cell strainer over a 50 milliliter conical tube and transfer the harvested organs onto the strainer. Using the plunger from a sterile three milliliter syringe mash the tissue through the strainer keeping the filter and organs moist throughout the maceration process with the addition of MS buffer as necessary. Next, rinse the strainer with five to 10 milliliters of MS buffer, followed by centrifugation of the resulting cell suspension.
Resuspend the pellet in five milliliters of ACK red blood cell lysis buffer. After five minutes on ice restore the tenacity with 10 to 20 milliliters of MS buffer and centrifuge the cells again. At the end of the centrifugation place a 30 micron cell strainer over a 15 milliliter conical tube and prime the strainer with one milliliter of MS buffer.
Resuspend the cells in five milliliters of fresh MS buffer and filter the cell suspension through the strainer to removed the lysed red blood cell aggregates. Add four milliliters of fresh MS buffer to rinse the empty lysis tube and pass the wash through the strainer. Count the cells and aliquot an appropriate number of splenocytes for the extracellular flux assay.
Then pellet both sets of cells, re suspending the splenocyte sample for the extracellular flux assay in five milliliters of RF 10 medium on ice. Re suspend the cells for bead isolation in the appropriate volumes of biotin antibody cocktail, anti biotin microbeads, and MS buffer for the indicated incubation times at two to eight degrees Celsius. At the end of the second incubation collect the cells by centrifugation and re suspend the pellet in 500 microliters of fresh MS buffer per one times 10 to the eight cells.
Next load the appropriate column onto the separation magnet, and prime the column with three milliliters of MS buffer, discarding the flow through. Place a sterile 15 milliliter conical tube under the column and load the column with the entire cell sample volume. Then rinse the cell incubation tube with three milliliters of fresh MS buffer collecting the wash in the conical tube.
After the last elute has been collected fill the tube to a final volume of 15 milliliters of MS buffer and centrifuge the negatively isolated cells. Then re suspend the pellet in five milliliters of fresh RF 10 medium on ice. No more than three hours after the isolation centrifuge all of the cell samples, and re suspend the pellets in five milliliters of the appropriate assay medium for another centrifugation.
After the second centrifugation re suspend the pellets at the appropriate experimental concentration to achieve a final volume of 180 microliters of cells per well in fresh assay medium and plate 180 microliters of cells into each well of the plate. Incubate the cells at 37 degrees Celsius for 25 minutes. Then centrifuge the plate to securely adhere the cells to the bottom of the wells, and return the plate to the incubator.
After 30 minutes use a micro pipette to load the freshly prepared 37 degree Celsius compounds of interest into the appropriate injector ports of the extracellular flux analyzer sensor cartridge, loading any control background and non experimental ports with medium alone. Return the loaded cartridge to the incubator and set up the extracellular flux assay program. Then load the cartridge and begin the program replacing the calibrant plate with the assay plate after the calibration as prompted.
After the extracellular flux assay proceed with the protein content measurements. Over 90%of all the cell populations are viable with a 98 to 99%purity observed for the lymphocytes. The confluence of each cell type correlates with the initial plating densities.
The lysate protein concentrations linearly correlate with the plating densities, confirming that protein concentrations can be used to accurately normalize extracellular flux data. Higher cell numbers correlate with the higher measured oxygen consumption rate, as well as with more dramatic responses to mitochondrial inhibitors. Higher plating densities result in similar normalized oxygen consumption rate measurements in T cells and splenocytes while five and 1.25 times 10 to the five cells per well result in similar normalized oxygen consumption rate measurements in B cells.
Mitochondrial respiration parameters also linearly correlate with the plating densities. Additionally the low proton leak indicates that most of the baseline respiration is used toward ATP synthesis. Higher plating densities result in higher extracellular acidification rates and greater responses to mitochondrial inhibitors.
Glycolysis parameters linearly correlate with the cell plating densities, with the high plating density determined to be the most optimal for a successful glycolysis stress assay. Glucose slightly stimulates mitochondrial respiration which is inhibited by oligomycin while 2-DG does not significantly affect the oxygen consumption. Once mastered this technique can be completed in six to eight hours if it is performed properly.
While attempting this procedure it's important to remember to keep the cells on ice prior to the extracellular flux analysis and to accurately count and plate the cells. Following this procedure other methods like measuring the mitochondrial potential and glucose uptake can be performed to answer additional questions regarding the metabolic state of the cells. After watching this video you should have a good understanding of how to isolate lymphocytes and to perform an extracellular flux assay.
