Name: Investigating Glycolysis in Primary Microglia Using Extracellular Flux Assay (Video) | JoVE Journal
Uploaded: 2026-04-10T12:45:00.000+00:00
Duration: 7 min 21 s
Description: Learn to measure glycolysis in primary microglia using an extracellular flux analyzer with glycolysis stress test. This protocol enables real-time ECAR assessment for studying metabolic changes in neuroinflammation.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The work is funded through CIHR project grant to DKK. The RTI to Dr. Craig Moore for Seahorse assay XFe24 analyzer is greatly acknowledged.

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
<ol>
	<li>Prepare dissection solution by adding 10 mM HEPES to 1x HBSS without Ca2+ and Mg2+.</li>
	<li>Prepare digestion solution (per 2&#8211;3 brains) by combining 1 mL of 2.5% trypsin (10x stock, undiluted), 2 mL of dissection solution, and 200 &#181;L DNase I (1 mg/mL).</li>
	<li>Prepare mixed glia medium by supplementing DMEM with 10% FBS, 1x Glutamax, and 1x antibiotic/antimycotic.</li>
	<li>Prepare microglia medium by supplementing DMEM with 10% FBS, 1x Glutamax, 1x MEM NEAA, 1x sodium pyruvate, and 1x penicillin/streptomycin.</li>
</ol>
2. Plating for mixed glia cultures (Figure 2A) 
<ol>
	<li>On day 1:
	<ol>
		<li>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.</li>
		<li>Decant the dissection solution and replace it with 3 mL of digestion solution per 2&#8211;3 brains. Gently triturate using a 2 mL serological pipette to dissociate the tissue.</li>
		<li>Place the tubes on a rotor within an incubator at 37 &#176;C, and mix by triturating using a 2 mL serological pipette every 8 min for a total of about 24 min.</li>
		<li>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.</li>
		<li>Using an 18 G needle in a 5 mL syringe, triturate the cells, then pass them through a 70 &#181;m nylon cell strainer. Centrifuge the collected cells again at 300 x g for 5 min at RT.</li>
		<li>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&#8211;3 brain cortices per flask is advised) 
		NOTE: Coating the flask with Poly-D-Lysine or another reagent is not required.</li>
	</ol>
	</li>
	<li>On day 2, change the whole media and add fresh mixed glia media.</li>
	<li>On day 3, replace about 2/3rd 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.</li>
	<li>On day 6, replace 2/3rd of the media with fresh mixed glia media. Confirm the presence of a confluent astrocyte layer.</li>
</ol>
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).
<ol>
	<li>Shake the flask at 250 rpm for 2 h at 37 &#176;C. Collect the entire media containing the floating cells into a 15 mL conical tube and centrifuge at 300 x g for 10 min.</li>
	<li>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&#8211;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.</li>
	<li>Incubate the microplate at 5% CO2 for 30&#8211;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).</li>
	<li>Exclude the four wells designated as &#8216;background&#8217; 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.</li>
</ol>
4. Treatment of primary microglia with pro-inflammatory stimuli
<ol>
	<li>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.</li>
</ol>
5. Preparation for the extracellular flux assay one day prior to running the experiment
<ol>
	<li>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 &#176;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).</li>
	<li>Prepare Glucose and 2DG stock solutions
	<ol>
		<li>Glucose &#8211; 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.</li>
		<li>2DG &#8211; Dissolve 328 mg in 2 mL of extracellular flux assay media. 
		&#8203;NOTE: Store both the glucose and 2DG at 4 &#176;C overnight</li>
	</ol>
	</li>
	<li>Aliquot 30-40 mL of assay media in a 50mL conical tube and store at 4 &#176;C for performing the assays the next day.</li>
</ol>
6. Extracellular Flux Assay (Day 12)
<ol>
	<li>On day 12, warm up the pre-constituted glucose, 2DG, and the aliquoted assay media at 37 &#176;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 &#181;L, then 10 &#181;L).</li>
	<li>Vortex briefly for uniform mixing until the pH reaches above 7. 
	NOTE: pH indicator strips are recommended for consistent pH monitoring.</li>
	<li>Add 2 &#181;L of stock solution of 10mM oligomycin in 2 mL of pH-adjusted assay media.
	<ol>
		<li>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 &#176;C until further use.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Collect the supernatant (discard the pellet to get rid of cells/debris) and store it in -80 &#176;C for lactate measurements, mentioned in Step 8.</li>
	<li>Wash the cells with 1 mL of pH-adjusted assay media very gently to avoid lifting off the cells.</li>
	<li>Discard the assay media and add 500 &#181;L of pH-adjusted assay media and incubate at 37 &#176;C (without CO2) for 45 min to 1 h.</li>
	<li>In the meantime, add all the required inhibitors to the extracellular flux analyzer&#8217;s calibration plate in their respective ports- 55 &#181;L of glucose to port A on the top left in all the wells, 60 &#181;L of oligomycin in port B to the top right wells and 65 &#181;L 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.</li>
	<li>After adding the inhibitors, set up a glycolysis stress test using the Agilent Seahorse Wave software (refer to&#160;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.</li>
	<li>Once the calibration is over, the machine will prompt to unload the calibration plate and load the culture plate.
	<ol>
		<li>Step I: Select Assay Template (Figure 3A)
		<ol>
			<li>Open the Wave software, and from the Templates section, choose the appropriate assay template for the experiment.</li>
			<li>In this case, select &#8220;XF Glycolysis Stress Test&#8221; on the extracellular flux analyzer.</li>
		</ol>
		</li>
		<li>Step II: Define Groups and Injection Strategies (Figure 3B)
		<ol>
			<li>Go to the Group Definitions tab and create experimental groups (e.g., Background, Control, and LPS).</li>
			<li>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).</li>
		</ol>
		</li>
		<li>Step III: Assign Groups to Plate Map (Figure 3C)
		<ol>
			<li>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.</li>
			<li>Ensure blank/background wells are designated for accurate normalization.</li>
		</ol>
		</li>
		<li>Step IV: Set Up the Protocol (Figure 3D)
		<ol>
			<li>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) &#8594; measures glycolysis, Injection of Oligomycin (Port B) &#8594; measures glycolytic capacity, Injection of 2-DG (Port C) &#8594; measures glycolytic reserve</li>
			<li>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&#39;s default settings.</li>
			<li>Review the Total Assay Time before running the assay.</li>
		</ol>
		</li>
		<li>Step V: Normalization (Figure 3E)
		<ol>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
7. Collection of cells for data quantification and normalization 
<ol>
	<li>Once the extracellular flux assay is complete, remove the culture plate and either proceed with protein extraction immediately or store the plate at -80 &#176;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).</li>
	<li>For immediate protein isolation, remove the assay media from the wells and wash once with 500 &#181;L of RT PBS (1x) (a gentle one-wash is advised to minimize disruption of the cell monolayer).</li>
	<li>To collect cells that may detach during this process, centrifuge the plate once at 300 &#215; g for 5 min, discard the PBS, and resuspend the resulting pellet in RIPA buffer to ensure accurate total protein measurements.</li>
	<li>Add about 75&#8211;100 &#181;L 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.</li>
	<li>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 &#176;C.</li>
	<li>Centrifuge the cell lysate at 16,000 x g for 15 min at 4 &#176;C. Discard the pellet as it will contain cellular debris.</li>
	<li>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 &#176;C (for short-term storage), or -80 &#176;C (for long-term storage).</li>
	<li>When restarting after storing the samples at either -20 &#176;C or -80 &#176;C, thaw them on ice for about 20&#8211;30 min, briefly vortex/spin, and continue with the standard BCA protocol.</li>
	<li>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&#8211;200 &#181;L of trypsin (pre-warmed to 37 &#176;C) immediately after the assay is complete and the assay media is removed, and incubate at 37 &#176;C for 5 min. Collect the cells by adding 100&#8211;200 &#181;L of 10% FBS to neutralize the effects of trypsin.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Generate the results and graph using the option &#8220;Export&#8221; followed by &#8220;Graph Pad Prism&#8221; option and &#8220;Excel sheet&#8221; 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. </li>
</ol>
8. Lactate measurements from culture supernatants
<ol>
	<li>Gradually thaw the supernatant aliquots on ice that were collected on day 12 and stored at &#8722;80 &#176;C from Step 6.5.</li>
	<li>Determine L-lactate concentrations using the L-Lactate Assay Kit in fluorometric mode following the manufacturer&#39;s protocol. 
	Note: The fluorometric mode is much more sensitive than the colorimetric assay.</li>
	<li>Prepare standards by serial dilution of the provided lactate standard to generate a calibration curve ranging from 0 to 0.1 nmol/well.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

<ol><li>Prinz, M., Jung, S., Priller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+biology%3A+One+century+of+evolving+concepts%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Microglia biology: One century of evolving concepts.</a> Cell. 179, 292-311 (2019).</li>
<li>Tay, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unique+microglia+recovery+population+revealed+by+single-cell+RNAseq+following+neurodegeneration%5BTitle%5D)+AND+%22Acta+Neuropathol.+Commun%22%5BJournal%5D)">Unique microglia recovery population revealed by single-cell RNAseq following neurodegeneration.</a> Acta Neuropathol. Commun. 6, 87(2018).</li>
<li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glycolysis+and+oxidative+phosphorylation+play+critical+roles+in+natural+killer+cell+receptor-mediated+natural+killer+cell+functions%5BTitle%5D)+AND+%22Front.+Immunol%22%5BJournal%5D)">Glycolysis and oxidative phosphorylation play critical roles in natural killer cell receptor-mediated natural killer cell functions.</a> Front. Immunol. 11, 202(2020).</li>
<li>Soto-Heredero, G., Gómezde Las Heras, M. M., Gabandé-Rodríguez, E., Oller, J., Mittelbrunn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glycolysis-a+key+player+in+the+inflammatory+response%5BTitle%5D)+AND+%22FEBS+J%22%5BJournal%5D)">Glycolysis-a key player in the inflammatory response.</a> FEBS J. 287, 3350-3369 (2020).</li>
<li>O'Neill, L. A., Kishton, R. J., Rathmell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+guide+to+immunometabolism+for+immunologists%5BTitle%5D)+AND+%22Nat.+Rev.+Immunol%22%5BJournal%5D)">A guide to immunometabolism for immunologists.</a> Nat. Rev. Immunol. 16, 553-565 (2016).</li>
<li>Bernier, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglial+metabolic+flexibility+supports+immune+surveillance+of+the+brain+parenchyma%5BTitle%5D)+AND+%22Nat.+Commun%22%5BJournal%5D)">Microglial metabolic flexibility supports immune surveillance of the brain parenchyma.</a> Nat. Commun. 11, 1559(2020).</li>
<li>Bernier, L. P., York, E. M., MacVicar, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunometabolism+in+the+brain%3A+How+metabolism+shapes+microglial+function%5BTitle%5D)+AND+%22Trends+Neurosci%22%5BJournal%5D)">Immunometabolism in the brain: How metabolism shapes microglial function.</a> Trends Neurosci. 43, 854-869 (2020).</li>
<li>Geric, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolic+reprogramming+during+microglia+activation%5BTitle%5D)+AND+%22Immunometabolism%22%5BJournal%5D)">Metabolic reprogramming during microglia activation.</a> Immunometabolism. 1, e190002(2019).</li>
<li>Yoo, I., Ahn, I., Lee, J., Lee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+flux+assay+%28Seahorse+assay%29%3A+Diverse+applications+in+metabolic+research+across+biological+disciplines%5BTitle%5D)+AND+%22Mol.+Cells%22%5BJournal%5D)">Extracellular flux assay (Seahorse assay): Diverse applications in metabolic research across biological disciplines.</a> Mol. Cells. 47, 100095(2024).</li>
<li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glutamine+metabolism+modulates+microglial+NLRP3+inflammasome+activity+through+mitophagy+in+Alzheimer%E2%80%99s+disease%5BTitle%5D)+AND+%22J.+Neuroinflammation%22%5BJournal%5D)">Glutamine metabolism modulates microglial NLRP3 inflammasome activity through mitophagy in Alzheimer’s disease.</a> J. Neuroinflammation. 21, 261(2024).</li>
<li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucose+transporter+1+critically+controls+microglial+activation+through+facilitating+glycolysis%5BTitle%5D)+AND+%22Mol.+Neurodegener%22%5BJournal%5D)">Glucose transporter 1 critically controls microglial activation through facilitating glycolysis.</a> Mol. Neurodegener. 14, 2(2019).</li>
<li>Lu, J., Zhou, W., Dou, F., Wang, C., Yu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TRPV1+sustains+microglial+metabolic+reprogramming+in+Alzheimer%27s+disease%5BTitle%5D)+AND+%22EMBO+Rep%22%5BJournal%5D)">TRPV1 sustains microglial metabolic reprogramming in Alzheimer's disease.</a> EMBO Rep. 22, e52013(2021).</li>
<li>Orihuela, R., McPherson, C. A., Harry, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglial+M1%2FM2+polarization+and+metabolic+states%5BTitle%5D)+AND+%22Br.+J.+Pharmacol%22%5BJournal%5D)">Microglial M1/M2 polarization and metabolic states.</a> Br. J. Pharmacol. 173, 649-665 (2016).</li>
<li>Cheng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+glycolytic+reprogramming+controls+microglial+inflammatory+activation%5BTitle%5D)+AND+%22J.+Neuroinflammation%22%5BJournal%5D)">Early glycolytic reprogramming controls microglial inflammatory activation.</a> J. Neuroinflammation. 18, 129(2021).</li>
<li>Kaushik, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+glycolytic+metabolism+supports+transmigration+of+brain-infiltrating+macrophages+in+multiple+sclerosis%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">Enhanced glycolytic metabolism supports transmigration of brain-infiltrating macrophages in multiple sclerosis.</a> J. Clin. Invest. 129, 3277-3292 (2019).</li>
<li>Hu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((mTOR-mediated+metabolic+reprogramming+shapes+distinct+microglia+functions+in+response+to+lipopolysaccharide+and%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">mTOR-mediated metabolic reprogramming shapes distinct microglia functions in response to lipopolysaccharide and.</a> Glia. 68, 1031-1045 (2020).</li>
<li>Montilla, A., Zabala, A., Matute, C., Domercq, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+and+metabolic+characterization+of+microglia+culture+in+a+defined+medium%5BTitle%5D)+AND+%22Front.+Cell.+Neurosci%22%5BJournal%5D)">Functional and metabolic characterization of microglia culture in a defined medium.</a> Front. Cell. Neurosci. 14, 22(2020).</li>
</ol>

The authors have no conflict of interest to declare.

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&#8211;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...

Cells in the central nervous system change how they produce energy in response to normal and disease conditions1,2. 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, respectively3,4. 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 flexibility6,7. 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 CO&#8322; 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 &#8216;glycolysis&#8217;. 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&#39;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 bioenergetics10,11,12,13. ECAR assays and OCR assays are thus performed under different conditions.

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

investigating glycolysis in primary microglia using extracellular flux assay

Investigating Glycolysis in Primary Microglia Using Extracellular Flux Assay

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.

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<strong...

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.

Microglia, the resident macrophage cells of the central nervous system, dynamically alter their metabolic programs in response to physiological and ...

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.

investigating-glycolysis-primary-microglia-using-extracellular-flux

Research

JoVE Journal

Immunology and Infection

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions.  This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

This protocol provides a biochemical approach for measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of primary leukemic cells with extracellular flux assay. The assessment of the metabolic profile of these cells helps to characterize their demands, leading to personalized therapy.

Extracellular Flux Assay: A Method for Measuring Metabolic Profile of Cells

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage’s metabolism and phenotype.

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

In this paper, we describe a method to measure glycolysis and mitochondrial respiration in primary human Natural Killer (NK) cells isolated from peripheral blood, at rest or following IL15-induced activation. The protocol described could be easily extended to primary human NK cells activated by other cytokines or soluble stimuli.

Analysis of Human Natural Killer Cell Metabolism

Here we present two protocols to quantify microglial engulfment of vGLUT1-positive synapses and pHRodo Red-labeled crude synaptosomes using flow cytometry.

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Alcohol misuse impairs alveolar macrophage (AM) immunity due to suppressed mitochondrial respiration and bioenergetics. We recently demonstrated that ethanol (EtOH) exposure increases glutamine dependency for mitochondrial respiration in AMs. Herein, methods are provided to determine the usage of glutamine for mitochondrial respiration in EtOH-treated AMs using an extracellular flux bioanalyzer.

Assessment of Glutamine as a Fuel Source for Alveolar Macrophages Exposed to Chronic Ethanol Using an Extracellular Flux Bioanalyzer

Here, we describe in detail methods for extracting macrophages from the bone marrow, spleen, and infarcted heart, and subsequently assessing metabolic flux in live cells.

Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

A protocol was developed for the preparation of purified mitochondria from microglial cells, isolation of mitochondrial proteins for N-glycan release, and rapid detection of subcellular, mitochondrial glycans using infrared matrix-assisted laser desorption electrospray ionization coupled to high-resolution accurate mass analyzer mass spectrometry.

Mitochondrial Preparation from Microglia for Glycan Analysis

Learn to measure glycolysis in primary microglia using an extracellular flux analyzer with glycolysis stress test. This protocol enables real-time ECAR assessment for studying metabolic changes in neuroinflammation.

Investigating Glycolysis in Primary Microglia Using Extracellular Flux Assay

Explore More Videos