Investigating Glycolysis in Primary Microglia Using Extracellular Flux Assay

This research focuses on investigating glycolysis in primary microglia during inflammation, using extracellular flux assays with lab prepared metabolic inhibitors. Existing methods utilize expensive kits and lack sufficient detail. This protocol instead offers comprehensive, step-by-step guidance for affordable, reproducible results.

To begin, obtain 10-day-old mixed glial cultures derived from the cerebral cortices of postnatal day zero to two C57 black 6C mouse pups. Shake the flask at 250 revolutions per minute for two hours at 37 degrees Celsius. Collect the entire media containing the floating cells into a 15 milliliter conical tube.

Centrifuge at 300 G for 10 minutes. Then re-suspend the pellet in microglial media. In the cell culture microplate of the extracellular flux analyzer Mark four wells with one well in each row.

As background controls, add assay medium to the background control wells. Plate around 250, 000 cells per well in the remaining wells. Incubate the microplate at 5%carbon dioxide for 30 to 45 minutes, then replace the media with fresh microglia media and continue incubation.

To treat the cells on day 11, first assign the control and lipopolysaccharide, or LPS wells, randomly to rule out positional effects. Then treat the cells with 100 nanograms per milliliter LPS for 24 hours. One day before the assay add one milliliter calibrant buffer to all wells of the 24 well calibration plate.

Keep the plate overnight at 37 degrees Celsius without carbon dioxide. Prepare the glucose and 2-DG stock solutions and store both solutions at four degrees Celsius overnight. On the next day, aliquot 30 to 40 milliliters of assay media in a 50 milliliter conical tube for the assays.

Warm the stock solutions and the aliquoted assay media at 37 degrees Celsius. Add one normal sodium hydroxide in increments and vortex briefly to adjust the pH of each solution to around 7.4. Add oligomycin stock solution to two milliliters of pH-adjusted assay media.

Then remove the cells from the incubator, aspirate the media and gently wash them with one milliliter of the pH-adjusted assay media to prevent detachment. Discard the wash media and add 500 microliters of pH-adjusted assay media. Incubate at 37 degrees Celsius without carbon dioxide for 45 minutes to one hour.

Add all the required inhibitors to the extracellular flux analyzer's calibration plate in their respective ports. Load all wells, including background control wells, then remove the top cover from the plate and the pink cover between the plates. Place the calibration plate into the real-time extracellular flux analyzer.

Start the run using the Wave desktop platform to calibrate the instrument. Unload the calibration plate and load the culture plate when prompted by the machine. Open the Wave software and select the appropriate assay template from the template section.

Choose XF glycolysis stress test on the extracellular flux analyzer. Open the group definitions tab and create the experimental groups, like background, control, and LPS. Define the injection strategy by assigning glucose, oligomycin, and 2-DG to the corresponding ports.

Now, open the plate map view and assign wells to each group by dragging the group names onto the plate layout. Designate the blank or background wells for accurate normalization. Open the protocol tab and configure the measurement cycles and injections for non glycolytic acidification, followed by glucose, oligomycin, and 2-DG injections.

Define the number of measurement cycles after each injection and adjust the timing or use the software default settings. Review the total assay time before running the assay. Use the built-in normalization option to normalize the data.

Compared to the untreated control, LPS-treated primary microglia exhibited slight changes in morphology after 24 hours. LPS-treated primary microglia showed increased extracellular acidification rate over time compared to control microglia in the glycolysis stress test profile, and in the protein-normalized extracellular acidification rate profile. LPS-treated microglia demonstrated significantly increased glycolysis and glycolytic capacity compared to untreated controls, as indicated by elevated ECAR levels.

The ECAR values normalized to their protein content exhibited similar trends. Moreover, no significant differences were observed in glycolytic reserve between control and LPS-treated microglia with and without normalization. This protocol measures extracellular acidification rate to quantify basal glycolysis, glycolytic capacity, and real-time metabolic changes in cultured microglia, precise pH control, optimal cell density, gentle handling and data normalization are critical to avoid variability and inaccurate extracellular acidification rate measurements.

This protocol focuses on glycolysis measurement and additionally allows protein content or cell density analysis for normalization after the assay.