Investigating Glycolysis in Primary Microglia Using Extracellular Flux Assay

Summary

This protocol describes a cost-effective way to perform a glycolysis stress test to quantify metabolic reprogramming in primary mouse microglia. After isolation and inflammatory stimulation, extracellular acidification rate (ECAR) is measured using freshly prepared, pH-adjusted reagents, followed by protein normalization to enable accurate and reproducible assessment of glycolytic shifts.

Abstract

Microglia, the resident macrophage cells of the central nervous system, dynamically alter their metabolic programs in response to physiological and pathological cues. Understanding these metabolic shifts is crucial for elucidating their roles in inflammation. Here, we present a detailed protocol for assessing the glycolytic profile of primary microglia isolated from neonatal mouse brain cortices using a glycolysis stress test on an extracellular flux analyzer. This assay enables real-time measurement of ECAR, an indicator of glycolytic activity associated with low pH, such as lactate. Our approach involves treating cultured microglia under different conditions to examine how metabolic pathways are altered in response to various stimuli, including pro-inflammatory stimuli. Uniquely, our lab prepares fresh stock solutions of different reagents, including glucose, oligomycin, and 2-deoxyglucose (2DG), to target the different aspects of the pathway, with careful adjustment of pH for each reagent to ensure experimental accuracy and reproducibility. This method provides a robust platform for investigating glycolysis in primary microglia and offers insight into their functional states under inflammatory or disease-relevant conditions.

Introduction

Cells in the central nervous system change how they produce energy in response to normal and disease conditions^1,2. Among major metabolic pathways, glycolysis and oxidative phosphorylation (OXPHOS) are key pathways that regulate immune cell functions- they undergo either to perform a robust and immediate reaction to pro-inflammatory and anti-inflammatory conditions, respectively^3,4. Thus, immune cells can switch between metabolic pathways in response to inflammatory signals and functional states⁵. Microglia, like their myeloid counterparts, are metabolically tied to their functions and rapidly adapt to different pathways as an alternate metabolic fuel, demonstrating metabolic flexibility^6,7. Microglia undergo rapid metabolic reprogramming during inflammation, with enhanced glycolysis serving as a key metabolic signature of pro-inflammatory states⁸. Thus, assays that identify cellular dependence on a metabolic phenotype allow us to investigate their functional states under various conditions. In the current protocol, we aim to determine whether primary microglia rely on increased glycolysis during pro-inflammatory conditions. For this, we perform glycolysis stress test assays routinely. While the Seahorse XFe24 analyzer (extracellular flux analyzer) has been widely used to measure ECAR in immortalized cell lines and transformed cells, primary microglia pose unique technical challenges, including limited cell yields, variable adherence properties, and high sensitivity to culture conditions, which can confound metabolic measurements. Moreover, it provides real-time measurement of ECAR, enabling precise evaluation of lactate-driven glycolysis at multiple time points, which cannot be captured by endpoint lactate assays alone. Extracellular flux-based metabolic profiling has been widely adopted in metabolism research to characterize cell bioenergetics, making this protocol well-suited for studies investigating metabolic shifts in cells during activation, disease modeling, or drug testing. Readers can determine suitability based on their need for dynamic, high-resolution glycolytic measurements within primary cells.

Glucose is metabolized to pyruvate via glycolysis and then either converted to lactate in the cytoplasm or to CO₂ and water in mitochondria. The conversion of glucose to lactate leads to the release of protons into the extracellular space, resulting in medium acidification. The extracellular flux analyzer quantifies this acidification as the ECAR⁹. In the glycolysis stress test, cells are first incubated in a glucose- and pyruvate-free medium, and their baseline ECAR is recorded. The first injection delivers a saturating amount of glucose, which cells metabolize via glycolysis to pyruvate, producing ATP, NADH, water, and protons. The increased proton release elevates ECAR, reflecting ‘glycolysis’. Next, oligomycin is added to inhibit mitochondrial ATP synthesis, forcing cells to rely solely on glycolysis. This change in the proton gradient further increases ECAR and is measured as the cell's maximum glycolytic capacity. Finally, 2DG, a glucose analog, is introduced. It competitively inhibits hexokinase, blocking glycolysis and reducing ECAR. This drop confirms that prior ECAR was glycolysis-dependent. The difference between the maximum glycolytic capacity and the level of glycolysis defines the glycolytic reserve. The initial ECAR before glucose addition represents non-glycolytic acidification from other cellular processes (Figure 1).

We performed a glycolysis stress test using the extracellular flux analyzer. Raw ECAR values were exported to Excel for analysis. For each condition, we identified the time points corresponding to basal ECAR (prior to glucose addition), glycolysis (after glucose injection), and glycolytic capacity (after oligomycin treatment). ECAR values from the relevant time points within each phase and across the four technical replicates were averaged, and glycolysis was calculated as the increase in ECAR following glucose addition. Glycolytic capacity was calculated as an increase in ECAR after oligomycin addition. Glycolytic reserve was calculated as a decrease in ECAR after 2DG injection. These average values were then used to compare metabolic responses between groups.

On the other hand, to assess oxygen consumption rate (OCR) and evaluate mitochondrial functions, mitochondrial stress test (Agilent) is the standard approach, which measures parameters such as basal respiration, ATP-linked respiration, proton leak, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration. These measurements are obtained by sequentially injecting specific mitochondrial inhibitors, oligomycin (ATP synthase inhibitor), FCCP (Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone), an uncoupler that drives maximal respiration, and rotenone/antimycin A (complex I/III inhibitors), allowing quantitative assessment of mitochondrial health and bioenergetics^10,11,12,13. ECAR assays and OCR assays are thus performed under different conditions.

Protocol

All animal procedures were conducted in accordance with the guidelines of the Canadian Council on Animal Care and received approval from the Animal Care Committee at Memorial University of Newfoundland under protocol number 22-01-DK.

1. Preparations prior to the start of the experiments

1. Prepare dissection solution by adding 10 mM HEPES to 1x HBSS without Ca²⁺ and Mg²⁺.
2. Prepare digestion solution (per 2–3 brains) by combining 1 mL of 2.5% trypsin (10x stock, undiluted), 2 mL of dissection solution, and 200 µL DNase I (1 mg/mL).
3. Prepare mixed glia medium by supplementing DMEM with 10% FBS, 1x Glutamax, and 1x antibiotic/antimycotic.
4. Prepare microglia medium by supplementing DMEM with 10% FBS, 1x Glutamax, 1x MEM NEAA, 1x sodium pyruvate, and 1x penicillin/streptomycin.

2. Plating for mixed glia cultures (Figure 2A)

1. On day 1:
   1. Collect postnatal day (P0)-P2 pups from C57BL/6C mice and extract the cortices using a stereo microscope, and remove the meninges. Then, dissect the cortices into tiny pieces in dissection solution and transfer them into a 15 mL conical tube. Allow the tissues to settle at the bottom of the tube.
   2. Decant the dissection solution and replace it with 3 mL of digestion solution per 2–3 brains. Gently triturate using a 2 mL serological pipette to dissociate the tissue.
   3. Place the tubes on a rotor within an incubator at 37 °C, and mix by triturating using a 2 mL serological pipette every 8 min for a total of about 24 min.
   4. Following the tissue digestions, centrifuge the cells at 300 x g for 5 min at room temperature (RT). Then, discard the supernatant and resuspend the pellet in 5 mL of mixed glia media.
   5. Using an 18 G needle in a 5 mL syringe, triturate the cells, then pass them through a 70 µm nylon cell strainer. Centrifuge the collected cells again at 300 x g for 5 min at RT.
   6. Resuspend the pellet in 1 mL of mixed glia media initially and triturate multiple times to remove cell clumping, and then again resuspend in a higher volume, making up to 12 mL in each T75 flask (2–3 brain cortices per flask is advised)
      NOTE: Coating the flask with Poly-D-Lysine or another reagent is not required.
2. On day 2, change the whole media and add fresh mixed glia media.
3. On day 3, replace about 2/3^rd of the media with fresh mixed glia media. Allow the cells to grow for an additional seven days, for a total of 10 days.
   NOTE: At this point, an astrocyte layer can be observed.
4. On day 6, replace 2/3rd of the media with fresh mixed glia media. Confirm the presence of a confluent astrocyte layer.

3. Obtaining pure primary microglia (Figure 2A)

NOTE: Around day 10, small, round cells floating on top of the astrocyte layer are visible; these are predominantly microglia (Figure 2B).

1. Shake the flask at 250 rpm for 2 h at 37 °C. Collect the entire media containing the floating cells into a 15 mL conical tube and centrifuge at 300 x g for 10 min.
2. Resuspend the pellet in microglia media and plate around 250,000 cells per well in the cell culture microplates of the extracellular flux analyzer.
   NOTE: Around 3–4 million cells per mL are obtained from three T75 flasks; coating of the cell culture plates with poly-D-lysine or other reagents is not a requirement.
3. Incubate the microplate at 5% CO₂ for 30–45 min. After that, change the media again to obtain a pure microglia culture and to remove any oligodendrocytes that may still be floating. Cells are ready for treatment the very next day (Figure 2C).
4. Exclude the four wells designated as ‘background’ controls from cell seeding: one in each row (Figure 3C). These wells receive assay media.
   NOTE: Fill the unused wells with sterile water or PBS to prevent media evaporation, and plate the cells in a randomized pattern rather than in fixed rows or columns to minimize plate-based variability.

4. Treatment of primary microglia with pro-inflammatory stimuli

1. On day 11, treat the cells with 100 ng/mL lipopolysaccharide (LPS) for 24 hours. Randomize control and LPS-treated wells to rule out any positional effects.
   NOTE: Treatment durations may vary depending on the experimental conditions and need prior optimization.

5. Preparation for the extracellular flux assay one day prior to running the experiment

1. On day 11, add 1 mL calibrant buffer to the calibration plate (24-well format; extracellular flux assay kit) in all the wells. Keep the plate overnight at 37 °C without CO₂.
   NOTE: The calibration plate consists of 4 injection wells. For the glycolysis stress test, glucose is added to Port A, oligomycin is added to Port B, and 2DG is injected into Port C (refer to Figure 4A).
2. Prepare Glucose and 2DG stock solutions
   1. Glucose – Dissolve 90 mg of glucose in 2mL of extracellular flux assay media (Table of Materials). Initially, dissolve in about 1.9 mL so that, when pH is adjusted the next day with 1N NaOH, the final volume does not exceed 2 mL, which is critical to maintain the molarity.
   2. 2DG – Dissolve 328 mg in 2 mL of extracellular flux assay media.
      ​NOTE: Store both the glucose and 2DG at 4 °C overnight
3. Aliquot 30-40 mL of assay media in a 50mL conical tube and store at 4 °C for performing the assays the next day.

6. Extracellular Flux Assay (Day 12)

1. On day 12, warm up the pre-constituted glucose, 2DG, and the aliquoted assay media at 37 °C. Adjust the pH of glucose and 2DG, and that of the aliquoted assay media, to 7.4 by adding 1N NaOH in increments (e.g., 5 µL, then 10 µL).
2. Vortex briefly for uniform mixing until the pH reaches above 7.
   NOTE: pH indicator strips are recommended for consistent pH monitoring.
3. Add 2 µL of stock solution of 10mM oligomycin in 2 mL of pH-adjusted assay media.
   1. Stock solution preparation of 10mM oligomycin: Reconstitute a 10mg vial of oligomycin in 1mL of sterile DMSO, making a concentration of 10 mg/mL, and store at -80 °C until further use.
4. Take out the cells from the incubator (from step 4) and collect the media in 1.5 mL micro centrifuge tubes and centrifuge at 300 x g for 5 min.
5. Collect the supernatant (discard the pellet to get rid of cells/debris) and store it in -80 °C for lactate measurements, mentioned in Step 8.
6. Wash the cells with 1 mL of pH-adjusted assay media very gently to avoid lifting off the cells.
7. Discard the assay media and add 500 µL of pH-adjusted assay media and incubate at 37 °C (without CO₂) for 45 min to 1 h.
8. In the meantime, add all the required inhibitors to the extracellular flux analyzer’s calibration plate in their respective ports- 55 µL of glucose to port A on the top left in all the wells, 60 µL of oligomycin in port B to the top right wells and 65 µL of 2DG in port C to the bottom left wells (including background wells) (refer to Figure 4A).
   NOTE: The volume increase at each step is required to maintain the molarity, as volumes increase with the prior addition of glucose and oligomycin.
9. After adding the inhibitors, set up a glycolysis stress test using the Agilent Seahorse Wave software (refer to Figure 3). Then, run the calibration plate once prompted.
   NOTE: Remove the top cover from the plate and the pink cover between the plates before placing the calibration plate into the machine. During this step, the pH and O₂ sensors will be tested, and QC checked.
10. Once the calibration is over, the machine will prompt to unload the calibration plate and load the culture plate.
    1. Step I: Select Assay Template (Figure 3A)
       1. Open the Wave software, and from the Templates section, choose the appropriate assay template for the experiment.
       2. In this case, select “XF Glycolysis Stress Test” on the extracellular flux analyzer.
    2. Step II: Define Groups and Injection Strategies (Figure 3B)
       1. Go to the Group Definitions tab and create experimental groups (e.g., Background, Control, and LPS).
       2. Define the Injection Strategy by selecting the compounds to be injected in each port (e.g., glucose, oligomycin, 2DG for glycolysis stress test; these settings are pre-set by the software as default, check accordingly).
    3. Step III: Assign Groups to Plate Map (Figure 3C)
       1. Open the Plate Map view and assign wells to each group by dragging the group names (e.g., Background = black, Control = green, LPS = red) onto the plate layout.
       2. Ensure blank/background wells are designated for accurate normalization.
    4. Step IV: Set Up the Protocol (Figure 3D)
       1. Under the Protocol tab, program the measurement cycles and injections. Typical sequence for the glycolysis stress test includes Non-glycolytic acidification, Injection of Glucose (Port A) → measures glycolysis, Injection of Oligomycin (Port B) → measures glycolytic capacity, Injection of 2-DG (Port C) → measures glycolytic reserve
       2. Define the number of measurement cycles after each injection and adjust timing (e.g., 3 cycles of 3 min mix, 2 min wait, measure for 3 min).
          NOTE: For our experiments, we use the software's default settings.
       3. Review the Total Assay Time before running the assay.
    5. Step V: Normalization (Figure 3E)
       1. Normalize data with protein concentration values obtained from bicinchoninic acid (BCA) assay within Wave software using the built-in normalization option (e.g., by protein content, cell number, or other available methods). Protein concentration measurements are shown in Table 1.
          NOTE: The protocol for isolating the protein for the BCA assay after ECAR measurement is described in the following Step 7.

7. Collection of cells for data quantification and normalization

1. Once the extracellular flux assay is complete, remove the culture plate and either proceed with protein extraction immediately or store the plate at -80 °C until ready for protein extraction.
   NOTE: Protein assays allow us to normalize values across different treatment conditions (they are one of the methods to normalize the data, not the only method).
2. For immediate protein isolation, remove the assay media from the wells and wash once with 500 µL of RT PBS (1x) (a gentle one-wash is advised to minimize disruption of the cell monolayer).
3. To collect cells that may detach during this process, centrifuge the plate once at 300 × g for 5 min, discard the PBS, and resuspend the resulting pellet in RIPA buffer to ensure accurate total protein measurements.
4. Add about 75–100 µL of RIPA buffer to each of the wells and place the plate on a shaker (low-mid rotation) for 30 min (RT). Then, transfer the contents to a fresh 1.5 mL microcentrifuge tube.
   NOTE: Triturate and mix well within the wells prior to transferring the RIPA buffer into the 1.5 ml microcentrifuge tube.
5. Incubate the microcentrifuge tube on a rotator for about 30 min at RT for proper lysis of cells. During this time, pre-cool the centrifuge to 4 °C.
6. Centrifuge the cell lysate at 16,000 x g for 15 min at 4 °C. Discard the pellet as it will contain cellular debris.
7. Collect the supernatant for protein quantification using the BCA assay (or any other preferred method). Either proceed immediately or store the lysates after removing all the assay media at -20 °C (for short-term storage), or -80 °C (for long-term storage).
8. When restarting after storing the samples at either -20 °C or -80 °C, thaw them on ice for about 20–30 min, briefly vortex/spin, and continue with the standard BCA protocol.
9. Alternatively, count the cells per well using a hemocytometer or through a flow cytometer and integrate into the normalization strategies. In that case, add about 100–200 µL of trypsin (pre-warmed to 37 °C) immediately after the assay is complete and the assay media is removed, and incubate at 37 °C for 5 min. Collect the cells by adding 100–200 µL of 10% FBS to neutralize the effects of trypsin.
   1. Centrifuge the cells at 300 x g for 5 min, remove the supernatant, and resuspend the pellet in about 1 mL of 10% FBS before counting using a hemocytometer. Alternatively, the cells can be fixed with 4% PFA for 15 min before proceeding with flow cytometry-based cell-counting.
      NOTE: If proceeding with cell counting right away, PFA-based fixation is not required.
10. Normalize ECAR values with protein concentration from Step 7.8 or cell counts from Step 7.9.1 in each well using the Wave software as in Step 6.10.5.1. The resulting values are the normalized values for the glycolysis stress test assay.
11. Generate the results and graph using the option “Export” followed by “Graph Pad Prism” option and “Excel sheet” for each data point value for quantification.
    NOTE: In the cell culture plate, the choice of four designated blank wells is flexible and can be set by the experimenter. The default blank arrangement is shown in Figure 3C.

8. Lactate measurements from culture supernatants

1. Gradually thaw the supernatant aliquots on ice that were collected on day 12 and stored at −80 °C from Step 6.5.
2. Determine L-lactate concentrations using the L-Lactate Assay Kit in fluorometric mode following the manufacturer's protocol.
   Note: The fluorometric mode is much more sensitive than the colorimetric assay.
3. Prepare standards by serial dilution of the provided lactate standard to generate a calibration curve ranging from 0 to 0.1 nmol/well.
4. Add samples and standards to a clear 96-well microplate in duplicate, then add the reaction mixture containing lactate enzyme mix and fluorescent probe to each well.
5. Incubate at RT for 30 min protected from light. Measure fluorescence intensity using a fluorescence microplate reader with excitation/emission wavelengths set at 535/587 nm.
6. Calculate lactate concentrations in samples from the standard curve and express them as pM. Lactate levels from supernatants of untreated (control) and LPS-treated primary microglia are presented in Table 2.

Results

The extracellular flux-based glycolysis stress test successfully measures the glycolytic profile of primary microglia. The assay measures basal glycolysis, i.e., glycolysis after glucose injection, glycolytic capacity after oligomycin injection, and inhibition of glycolysis upon 2DG injection. For our experiment, we compared control (untreated) and LPS-treated primary microglia. We observed slight morphological changes between control and LPS-treated microglia post 24 hours (Figure 2D

Discussion

Microglial response during neuroinflammation is accompanied by profound metabolic rewiring that enables these cells to meet the energetic and biosynthetic demands of their functional state. The extracellular flux–based glycolysis stress test offers a sensitive and real-time method to measure glycolytic flux, enabling us to dissect how primary microglia adapt their metabolism under pro-inflammatory conditions. In this study, we utilized primary murine microglia and assessed their glycolytic activity following exposu...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1M HEPES solution	Sigma Aldrich	H0887
1X HBSS	Gibco	14175-079
2.5% Trypsin	Gibco	15090-046
2DG	Millipore sigma	D6134
Antibiotic/Antimycotic	Gibco	15240-062
D –(+)Glucose, anhydrous	Thermoscientific	A16828.36
DMEM	Gibco	11965-092
DNase I	Millipore sigma	11284932001- Roche
FBS	Fisherbrand	FB12999102
Glutamax	Gibco	35050-061
L-Lactate Kit	Abcam	ab65330
LPS	Sigma Aldrich	L4391
MEM NEAA	Gibco	11140-050
Oligomycin A	Millipore sigma	495455
Penicillin/Streptomycin	Gibco	15240-062
pH indicator strips	Fisherbrand	13-640-516
Seahorse XFe24	Agilent Technologies	N/A	Generic name: Extracellular flux analyzer
Seahorse 24 V7 PS cell culture microplates	Agilent Seahorse	100777-004	Generic name: Extracellular flux cell culture microplates
Seahorse assay XF Calibrant buffer (pH 7.4)	Agilent Seahorse	100840-000	Generic name: Extracellular flux calibrant buffer
Seahorse assay XF DMEM media (pH 7.4)	Agilent Seahorse	103575-100	Generic name: Extracellular flux assay media
Seahorse Extracellular flux assay kit	Agilent Seahorse	102342-100	Generic name: Extracellular flux calibration plate
Sodium pyruvate	Gibco	11360-070