Kasper M&#248;lgaard&#160;and Anne Rahbech received grants from T&#248;mmermester J&#248;rgen Holm og Hustru Elisa f. Hansens Mindelegat. Kasper M&#248;lgaardalso received a grant from B&#248;rnecancerfonden.

Experiments were carried out under the guidelines from Herlev Hospital and the Capital Region of Denmark.
NOTE: This protocol contains instructions for both an Optimization run and an Assays run. It is clearly written in the text when instructions are for an Optimization run or an Assay run. Run an Optimization run before continuing with the Assay runs
1. Human peripheral blood mononuclear (PBMC) isolation from buffy coats
<ol>
	<li>PBMC isolation
	<ol>
		<li>Collect buffy coats from the appropriate institution (collected in blood collection bags). Buffy coats originate from healthy donors. Exclude donors that have recently used painkillers.</li>
		<li>Spray the blood collection bag containing the buffy coat with 70% ethanol before transferring to a laminar flow cabinet. Always use sterile techniques and sterile hardware for all steps 
		NOTE: Ensure that the correct authorizations for the handling of human blood samples are obtained.</li>
		<li>Transfer the blood to a sterile 50 mL centrifuge tube.</li>
		<li>Dilute the blood at least 10% with non-supplemented RPMI 1640.</li>
		<li>Pour 20 mL of the preferred density gradient medium in a 50 mL centrifuge tube.</li>
		<li>Aspirate 25 mL of the diluted blood into a serological pipette. Set the electric pipette controller to the lowest speed.</li>
		<li>Hold the tube with density gradient medium at a 45&#176; angle and rest the serological pipette tip on the inner side of the 50 mL centrifuge tube. Slowly release 25 mL of the diluted blood from the serological pipette.</li>
		<li>Make sure the diluted blood and the density gradient medium do not mix. Ensure that the diluted blood rests on top of the density gradient medium.</li>
		<li>Repeat this with subsequent density gradient medium tubes until all diluted blood is processed.</li>
		<li>Carefully move the tubes to a centrifuge and centrifuge at 1000 x g for 30 min in a swing-out rotor at room temperature (RT). Ensure that the acceleration and break are at a minimum. 
		NOTE: After centrifugation, various layers should be visible. The top, clear pink-to-orange layer consists of the blood plasma and platelets. The middle white layer consists of the PBMCs followed by a clear layer that consists of the density gradient medium, and finally a dark red layer at the bottom with red blood cells.</li>
		<li>Using a sterile Pasteur pipette, carefully aspirate the PBMC-containing white layer to a 50 mL centrifuge tube. 
		NOTE: Make sure not to transfer the density gradient medium as this will affect downstream purification. Excess plasma will not affect the results.</li>
		<li>Pool all collected PBMCs in the same 50 mL centrifuge tube and top up to 50 mL with RPMI 1640.</li>
		<li>Centrifuge the cells at 500 x g for 5 min at RT (perform the remaining centrifugation steps at this setting unless specified otherwise).</li>
		<li>Aspirate the supernatant and resuspend the cells in 30 mL of RPMI 1640. Count the cells using a hemocytometer or automated cell counter.</li>
		<li>For cryopreservation, resuspend a maximum of 30 million cells per 1 mL of freezing medium.</li>
		<li>Transfer the cells to cryotubes and freeze until -80 &#176;C using a rate-controlled freezing container (-1 &#176;C per min). For long-term storage, move the PBMCs to -140 &#176;C. 
		&#8203;NOTE: Limit the time cells are in the freezing medium at RT. At RT, DMSO is highly toxic to cells.</li>
	</ol>
	</li>
</ol>
2. Culturing of activated human primary T lymphocytes
<ol>
	<li>Thawing of cells (Day 1)
	<ol>
		<li>Pre-warm 10 mL of RPMI 1640 per sample to around 37 &#176;C.</li>
		<li>Take the desired number of cell ampules and store them temporarily on dry ice.</li>
		<li>Resuspend the frozen cells in 10 mL of pre-warmed RPMI 1640.</li>
		<li>Centrifuge the cells for 5 min at 500 x g at RT.</li>
		<li>Discard the supernatant and wash the cells again by resuspending in 10 mL of RPMI 1640 and centrifuge as described in 2.1.4.</li>
		<li>Discard the supernatant and resuspend the cells at 2 x 106 cells per mL of X-VIVO 15 medium + 5% human serum (hereafter: T cell medium).</li>
		<li>Plate 2 mL of the cells per well in a 24-well cell culture plate and incubate overnight at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Activation of cells (Day 0)
	<ol>
		<li>Wash CD3/CD28 beads by transferring 12.5 &#181;L of beads per 1 million cells to a microcentrifuge tube. Add 12.5 &#181;L of PBS per 12.5 &#181;L of beads. 
		NOTE: It is important to vortex the vial of beads before use.</li>
		<li>Place the microcentrifuge tube on a suitable magnet for 1 min.</li>
		<li>Discard the buffer and resuspend the beads in the original volume of T cell medium (12.5 &#181;L of T cell medium per 12.5 &#181;L of the original volume of beads).</li>
		<li>Add 12.5 &#181;L of beads per million of cells corresponding to a ratio of 1:2 (beads: cells).</li>
		<li>Divide the cells into two conditions with around 5 million cells in each.</li>
		<li>Add the correct volume of cytokines to the conditions as mentioned in Table 1.</li>
		<li>Incubate the cells for 3 days at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Culturing of cells (Day 3 and 5)
	<ol>
		<li>Resuspend the cells and split them by transferring half the volume from each well into a new well. Add the same volume of fresh T cell medium to each well.</li>
		<li>Add new cytokines to each condition as mentioned in Table 1.</li>
	</ol>
	</li>
</ol>
3. Extracellular flux assay
<ol>
	<li>Hydration of the sensor cartridge (Day 0)
	<ol>
		<li>Unpack the sensor cartridge and carefully remove the sensor cartridge from the utility plate.</li>
		<li>Place the sensor cartridge upside-down, taking care not to touch the sensor probes.</li>
		<li>Fill the utility plate with 200 &#181;L of calibrant (see Table of Materials for details) and carefully replace the sensor cartridge back into the utility plate.</li>
		<li>Alternatively, to eliminate any bubble formation, incubate the sensor cartridge in sterile ultrapure water overnight and replace it with a pre-warmed calibrant on the morning of the assay.</li>
		<li>Incubate the sensor cartridge plate at 37 &#176;C in a non-CO2-regulated heating cabinet overnight. 
		&#8203;NOTE: It is very important to use a non-CO2 regulated cabinet since excess CO2 will influence the sensor cartridge. Make sure that the Flux analyzer (see Table of Materials for details) is turned on at least a day before use to allow it to warm up to 37 &#176;C.</li>
	</ol>
	</li>
	<li>Cell coating and preparation of mitochondrial inhibitors (Day 1)
	<ol>
		<li>Prepare a coating solution (see Table of Materials) containing NaHCO3 (pH 8.3, 0.1 M, 1128 &#181;L), Cell-Tak (1 mg/mL, 48 &#181;L) and NaOH (1.0 M, 24 &#181;L). 
		NOTE: Coating solution should always be made and used fresh.</li>
		<li>Open a fresh XF cell culture plate and add 12 &#181;L of the freshly prepared coating solution to each well. Ensure even distribution of coating solution in the bottom of all the wells.</li>
		<li>Incubate the plate at RT with the lid on for 30 min and discard the remaining liquid solution from all the wells.</li>
		<li>Wash the plate with 200 &#181;L of sterile water and discard the liquid.</li>
		<li>Wash the plate with 200 &#181;L of cell culture grade sterile PBS and discard the liquid.</li>
		<li>Leave the plate at RT and let it dry for at least 30 min.</li>
	</ol>
	</li>
	<li>Plate T cells in the XF cell culture plate (Day 1)
	<ol>
		<li>Prepare 50 mL of assay media by mixing suitable XF RPMI media with glucose, pyruvate, and glutamine according to experimental setup (Recommended levels: 4 mM glucose, 1 mM pyruvate and 3 mM glutamine).</li>
		<li>Heat to 37 &#176;C in a non-CO2 regulated incubator and set pH at 7.4. Ensure that there is enough media for plating cells and preparing oligomycin and FCCP solutions (section 3.4).</li>
		<li>Design a plate layout with an increasing number of cells per well for Optimization run or Assay run. Use four 4 wells, filled with media and injected with media, for background measurements.</li>
		<li>Count T cells prepared in section 2.3 (by the preferred method) and pipette the correct number of cells to each well of the XF cell culture plate coated with the coating solution, according to the plate layout. 
		&#8203;NOTE: The final volume of each well may vary but must be enough to cover the bottom of the well.</li>
		<li>Centrifuge the XF cell culture plate at 1000 x g at RT for 10 min to adhere the T cell to the coated surface.</li>
		<li>Wash the cells with 200 &#181;L of assay media, discard the media and add 180 &#181;L of assay media. 
		NOTE: Visually inspect the wells using an inverted light microscope to ensure that the cells are attached and evenly distributed across the well surface.</li>
		<li>Incubate the XF cell culture plate in the non-CO2 regulated heating cabinet for 30 min to ensure that the plate&#39;s temperature is 37 &#176;C.</li>
	</ol>
	</li>
	<li>Loading sensor cartridge with oligomycin and FCCP for Optimization run (Day 1) 
	NOTE: If oligomycin and FCCP concentrations were already optimized, continue at section 3.5.
	<ol>
		<li>Prepare working solutions of oligomycin and FCCP in assay media (prepared in step 3.3.1) as described in steps 3.4.2 and 3.4.3.</li>
		<li>Oligomycin working solutions: prepare 5 &#181;M solution (2 mL of assay media + 10 &#181;L of 1 mM oligomycin stock) and 3 &#181;M solution (8 mL of assay media + 24 &#181;L of 1 mM oligomycin stock).</li>
		<li>FCCP working solution: Prepare 2 &#181;M solution (2 mL of assay media + 13.2 &#181;L of 300 &#181;M FCCP stock) and a 1.3 &#181;M solution (8 ml of assay media + 34.6 &#181;L of 300 &#181;M FCCP stock)</li>
		<li>Load the working solutions of either oligomycin or FCCP into the injection ports of the sensor cartridge (Table 2). 
		NOTE. It is important that no injection ports contain air only. If, for any reason, not all injection ports are used, empty ports need to be filled with assay media.</li>
		<li>Gently knock the edges of the plate on the table to remove potential bubbles in the injection ports.</li>
	</ol>
	</li>
	<li>Loading sensor cartridge with oligomycin, FCCP, and antimycin A for Assay run (Day 1)
	<ol>
		<li>Prepare solutions of oligomycin, FCCP in assay media (prepared in step 3.3.1) according to the optimal concentrations identified in a previous Optimization run. Also, prepare a 20 &#181;M antimycin A solution.</li>
		<li>Load 20 &#181;L of either oligomycin or FCCP into injection port A of the sensor cartridge according to plate layout. Add 22 &#181;L of 20 &#181;M antimycin A to injection port B of all wells. The resulting concentration of antimycin A once injected into the well will be 2 &#181;M.</li>
	</ol>
	</li>
	<li>Setting up experimental protocol in Flux analyzer software.
	<ol>
		<li>Assign the groups and plate map for each condition per plate layout.</li>
		<li>Design the protocol according to the injection strategy (Table 3 and Figure 1). 
		NOTE: When running Assay runs, injection C and D can be omitted.</li>
		<li>Save the assay set up, fill out the information required to be included for the assay, and press Start.</li>
		<li>The flux analyzer will ask for the sensor cartridge prepared in section 3.5. Remove the lid and insert the sensor cartridge as directed by the analyzer. 
		NOTE: The assay will calibrate and check sensors indicated by checkmarks. After successful calibration, the analyzer will request for the XF cell culture plate prepared in section 3.3. The utility plate is ejected from the analyzer and replaced with the XF cell culture plate to start the assay.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>vander Windt, G. J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+respiratory+capacity+is+a+critical+regulator+of+CD8++T+cell+memory+development.">Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development.</a> Immunity. 36 (1), 68-78 (2012).</li><li>Krauss, S., Brand, M. D., Buttgereit, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+takes+a+breath--new+quantitative+perspectives+on+bioenergetics+and+signal+transduction.">Signaling takes a breath--new quantitative perspectives on bioenergetics and signal transduction.</a> Immunity. 15 (4), 497-502 (2001).</li><li>vander Windt, G. J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8+memory+T+cells+have+a+bioenergetic+advantage+that+underlies+their+rapid+recall+ability.">CD8 memory T cells have a bioenergetic advantage that underlies their rapid recall ability.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (35), 14336-14341 (2013).</li><li>Chang, C. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttranscriptional+control+of+T+cell+effector+function+by+aerobic+glycolysis.">Posttranscriptional control of T cell effector function by aerobic glycolysis.</a> Cell. 153 (6), 1239-1251 (2013).</li><li>vander Windt, G. J. W., Chang, C. -. H., Pearce, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+bioenergetics+in+T+cells+using+a+Seahorse+extracellular+flux+analyzer.">Measuring bioenergetics in T cells using a Seahorse extracellular flux analyzer.</a> Current Protocols in Immunology. 113, 1-14 (2016).</li><li>Buck, M. D., O'Sullivan, D., Pearce, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+metabolism+drives+immunity.">T cell metabolism drives immunity.</a> Journal of Experimental Medicine. 212 (9), 1345-1360 (2015).</li><li>Rivadeneira, D. B., Delgoffe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+T-cell+reconditioning:+Improving+metabolic+fitness+for+optimal+cancer+immunotherapy.">Antitumor T-cell reconditioning: Improving metabolic fitness for optimal cancer immunotherapy.</a> Clinical Cancer Research. 24 (11), 2473-2481 (2018).</li><li>Cieri, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-7+and+IL-15+instruct+the+generation+of+human+memory+stem+T+cells+from+naive+precursors.">IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors.</a> Blood. 121 (4), 573-584 (2013).</li><li>Kenwood, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+mitochondrial+uncoupler+that+does+not+depolarize+the+plasma+membrane.">Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane.</a> Molecular Metabolism. 3 (2), 114-123 (2013).</li><li>Alizadeh, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL15+enhances+CAR-T+cell+antitumor+activity+by+reducing+mTORC1+activity+and+preserving+their+stem+cell+memory+phenotype.">IL15 enhances CAR-T cell antitumor activity by reducing mTORC1 activity and preserving their stem cell memory phenotype.</a> Cancer Immunology Research. 7 (5), 759-772 (2019).</li></ol>

The authors declare no conflict of interest.

Detailed and correct quantification of oxidative phosphorylation is an indispensable tool when describing the energy states of T cells. The state of mitochondrial fitness can be directly related to T cell activation potential, survival, and differentiation1,5. With this protocol, it is possible to determine the various properties of oxidative phosphorylation (see Table 4 for a detailed explanation). Precise quantification of these properties of o...

Correct T cell development and function are essential for the ability of the immune system to recognize and respond to antigens. Mitochondrial oxidative phosphorylation (OxPhos) changes according to the state of the T cell. Na&#239;ve T cells predominantly use OxPhos to produce ATP, whereas activated T cells undergo a metabolic transition where glycolysis becomes dominant1. After the effector phase, the small remaining subset of memory T cells reverts to a metabolic state dominated by OxPhos2,3. The changes of OxPhos follow the differentiation of T cells to such a degree that even subsets of T cells can be differentiated by their specific is OxPhos properties1. Conversely, OxPhos is important for T cells&#39; function, and inhibition of OxPhos has been demonstrated to block proliferation and cytokine production of T cells4. Therefore, the ability to quantify the properties of T cell OxPhos in a precise and reproducible manner is a powerful tool for anyone working with T cells.
In this protocol, properties of T cell OxPhos are measured using an extracellular flux analyzer. The core function of this analyzer is to continuously measure the oxygen content of the growth media of the cells to be analyzed. Oxygen removed from the growth media is assumed to be taken up by the cells. By treating the cells with a variety of OxPhos inhibitors or modifiers, a drop in oxygen uptake is associated with the inhibited or modulated function. For example, inhibition of the ATP synthase will lead to a reduced cellular uptake of oxygen that would otherwise be used to produce ATP by oxidative phosphorylation. Other equipment, including the Clark electrode and the Oroboros instrument, offers similar functionality, and each instrument has different advantages and shortcomings. A wide array of cell types can be used for studies in these devices, but one particularly challenging cell type is human primary T lymphocytes5. Due to their small size, poor survival ex vivo, and non-adherent properties, human primary T cells can be challenging to study.
This is a protocol for studying the mitochondrial respiration of human primary T cells by an extracellular analyzer. The protocol is divided into an Optimization run, where optimal concentrations of cell number per well, as well as the optimal concentration of oligomycin and FCCP, are determined. Furthermore, an Assay run, where the optimized conditions are used.
Using blood-derived human PBMCs and ex vivo primary T cell cultures, this protocol demonstrates the importance of optimal inhibitor concentration and the relevance of using separate instead of a sequential injection of mitochondrial inhibitors when working with sensitive cell types. Finally, it is demonstrated that this assay can robustly detect subtle differences in mitochondrial respiration upon polarization with cytokines IL-2 and IL-15.

<table><tbody><tr><td>24-well tissue culture plate</td><td>Nunc</td><td>142485</td><td></td></tr><tr><td>Anti-CD3xCD28 beads</td><td>Gibco</td><td>11161D</td><td></td></tr><tr><td>Antimycin A</td><td>Merck</td><td>A8674</td><td></td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP)</td><td>Sigma-Aldrich</td><td>C2920</td><td></td></tr><tr><td>Cell-Tak</td><td>Corning</td><td>354240</td><td>For coating</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma Aldrich</td><td>D9170</td><td></td></tr><tr><td>Human Serum</td><td>Sigma Aldrich</td><td>H4522</td><td>Heat inactivated at 56 &amp;deg;C for 30 min</td></tr><tr><td>IL-15</td><td>Peprotech</td><td>200-02</td><td></td></tr><tr><td>IL-2</td><td>Peprotech</td><td>200-15</td><td></td></tr><tr><td>Lymphoprep</td><td>Stemcell Technologies</td><td>07801</td><td></td></tr><tr><td>Oligomycin</td><td>Merck</td><td>O4876</td><td></td></tr><tr><td>PBS</td><td>Thermo Fisher</td><td>10010023</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco-Thermo Fisher</td><td>61870036</td><td></td></tr><tr><td>Seahorse Calibrant</td><td>Agilent Technologies</td><td>102416-100</td><td></td></tr><tr><td>Seahorse XF 1.0 M glucose solution</td><td>Agilent Technologies</td><td>103577-100</td><td></td></tr><tr><td>Seahorse XF 100 mM pytuvate solution</td><td>Agilent Technologies</td><td>103578-100</td><td></td></tr><tr><td>Seahorse XF 200 mM glutamine solution</td><td>Agilent Technologies</td><td>103579-100</td><td></td></tr><tr><td>Seahorse XF RPMI medium, pH7.4</td><td>Agilent Technologies</td><td>103576-100</td><td>XF RPMI media</td></tr><tr><td>Seahorse XFe96 Analyser</td><td>Agilent Technologies</td><td></td><td>Flux analyzer</td></tr><tr><td>Seahorse XFe96 cell culture microplates</td><td>Agilent Technologies</td><td>102416-100</td><td>XF cell culture plate</td></tr><tr><td>Seahorse XFe96 sensor cartridge</td><td>Agilent Technologies</td><td>102416-100</td><td></td></tr><tr><td>Sodium Bicarbonate concentrate 0.1 M (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>36486</td><td></td></tr><tr><td>Sodium Hydroxide solution 1 N (NaOH)</td><td>Sigma Aldrich</td><td>S2770-100ML</td><td></td></tr><tr><td>X-VIVO 15</td><td>Lonza</td><td>BE02-060F</td><td></td></tr><tr><td>T cell beads magnet DynaMag-2 Magnet</td><td>Thermo Fisher</td><td>12321D</td><td></td></tr><tr><td>Seahorse wave</td><td></td><td></td><td>Flux analyzer software</td></tr></tbody></table>

real-time monitoring of mitochondrial respiration in cytokine-differentiated human primary t cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

A correct determination of OxPhos properties is an indispensable tool when studying T cells. However, if the assay conditions have not been optimized, there is a substantial risk of misleading or erroneous results. In this protocol, there is a strong focus on the optimization of cell number per well and concentrations of oligomycin and FCCP to be used. In the described setup, oligomycin and FCCP are added incrementally to the same well, increasing the concentration of the mitochondrial modulators. The optimal concentrati...

Watch this Scientific Journal Video about Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells at JoVE.com

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is ...

real-time-monitoring-mitochondrial-respiration-cytokine

Research

JoVE Journal

Immunology and Infection

3.8K Views.  University Hospital Herlev. Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented. 

Video: Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to objectively measure the severity of this condition. Here we describe an optimized method for assessing mitochondrial function of PBMCs collected from fatigued cancer patients. Using a compact extracellular flux system and sequential injection of respiratory inhibitors, we examined PBMC mitochondrial functional status by measuring basal mitochondrial respiration, spare respiratory capacity, and energy phenotype, which describes the preferred energy pathway to respond to stress. Fresh PBMCs are readily available in the clinical setting using standard phlebotomy. The entire assay described in this protocol can be completed in less than 4 hours without the involvement of complex biochemical techniques. Additionally, we describe a normalization method that is necessary for obtaining reproducible data. The simple procedure and normalization methods presented allow for repeated sample collection from the same patient and generation of reproducible data that can be compared between time points to evaluate potential treatment effects.

Our goal was to develop a practical protocol to evaluate mitochondrial dysfunction associated with fatigue in cancer patients. This innovative protocol is optimized for clinical use involving only standard phlebotomy and basic laboratory procedures.

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to ...

Cancer Research

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Medicine

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

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.

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and oxidative burst, and create neutrophil extracellular traps (NETs) for microbial clearance. New insights into the metabolic activities of neutrophils challenge the early concept that they primarily rely on glycolysis. Precise measurement of metabolic activities can unfold different metabolic requirements of neutrophils, including the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle), oxidative phosphorylation (OXPHOS), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) under physiological conditions and in disease states. This paper describes a step-by-step protocol and prerequirements to measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration on mouse bone marrow-derived neutrophils, human blood-derived neutrophils, and the neutrophil-like HL60 cell line, using metabolic flux analysis on a metabolic extracellular flux analyzer. This method can be used for quantifying the mitochondrial functions of neutrophils under normal and disease conditions.

We describe stepwise protocols measuring the mitochondrial respiration of mouse and human neutrophils and HL60 cells using the metabolic extracellular flux analyzer.

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Biology

Measuring Mitochondrial Function of Na&#239;ve and Effector CD8 T Cells

Understanding how immunometabolism impacts the function, differentiation, and fate of lymphocytes has garnered significant interest and attention. Lymphocyte biology has been explored using bioenergetic analysis and has now become a critically import tool in the field. Thus, we sought to optimize a bioenergetic analysis assay that can be adapted with pretreatments and acute injection for receptor stimulations. Here, we evaluated CD8 T cell ex vivo metabolism using the Cell Mito Stress Test to assess rates of oxygen consumption and extracellular acidification in na&#239;ve and effector CD8 T cells. Antigen-specific effector CD8 T cells were derived via ex vivo stimulation, and na&#239;ve CD8 T cells harvested from splenocytes and isolated with magnetic bead column separation. 
Pretreatments are performed in microplates and we detail how to prepare sensor cartridges. We show how injection ports can loaded with drugs to indirectly measure metabolic capacities and with metabolic modulators, this protocol can be used to study specific enzyme activity. T-cell receptor stimulations can be studied in real time with acute injection and stimulation with anti-CD3/CD28 using the injection ports. Instrument analyzers are used for measurements and data collection and data visualization is done with software programs to interpret cellular metabolism. This strategy produces an extensive amount of data on immune cell biology and mitochondrial bioenergetics allowing researchers to customize the protocol in numerous ways to explore CD8 T cell metabolism.

CD8 T cell bioenergetics can be interrogated using the Mito Stress Test. This methodology can be used to study acute and chronic metabolic programming. This protocol describes approaches to examine the relationships between T cell receptor biology and bioenergetic analysis.

Understanding how immunometabolism impacts the function, differentiation, and fate of lymphocytes has garnered significant interest and attention. ...

MATLAB Optimization for Analyzing Mitochondrial Networks in Neuroscience

Optimized Automated Analysis of Live Neuronal Mitochondria Homeostasis Modulation by Isoform-Specific Retinoic Acid Receptors

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in timelapse files, considerably simplifying and speeding up the process of image processing. Nonetheless, existing tools produce a large output volume, requiring individual manual attention, and basic experimental setups have an output of thousands of files, each requiring extensive and time-consuming handling. 
To address these issues, a routine optimization was developed, in both MATLAB code and live-script forms, allowing for swift file analysis and significantly reducing document reading and data processing. With a speed of 100 files/min, the optimization allows an overall rapid analysis. The optimization achieves the results output by averaging frame-specific data for individual mitochondria throughout time frames, analyzing data in a defined manner, consistent with those output from existing tools. Live confocal imaging was performed using the dye tetramethylrhodamine methyl ester, and the routine optimization was validated by treating neuronal cells with retinoic acid receptor (RAR) agonists, whose effects on neuronal mitochondria are established in the literature. The results were consistent with the literature and allowed further characterization of mitochondrial network behavior in response to isoform-specific RAR modulation. 
This new methodology allowed rapid and validated characterization of whole-neuron mitochondria network, but it also allows for differentiation between axon and cell body mitochondria, an essential feature to apply in the neuroscience field. Moreover, this protocol can be applied to experiments using fast-acting treatments, allowing the imaging of the same cells before and after treatments, transcending the field of neuroscience.

Author Spotlight: An Optimized Automated Method for Investigating Retinoic Acid Receptors in Neuronal Mitochondria

The mitochondrial network is extremely complex, making it very challenging to analyze. A novel MATLAB tool analyzes live confocal imaged mitochondria in timelapse images but results in a large output volume requiring individual manual attention. To address this issue, a routine optimization was developed, allowing for speedy file analysis.

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in ...

Neuroscience

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...

High-Resolution Analysis of Mitochondrial Function in Human PBMCs

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these changes&#160;do not always reflect what occurs in other tissues, such as skeletal muscle, these cells are an accessible and valuable source of viable mitochondria from human subjects. PBMCs are exposed to systemic signals that impact their bioenergetic state. Thus, expanding our tools to interrogate mitochondrial metabolism in this population will elucidate mechanisms related to disease progression. Functional assays of mitochondria are often limited to using respiratory outputs following maximal substrate, inhibitor, and uncoupler concentrations to determine the full range of respiratory capacity, which may not be achievable in vivo. The conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by ATP-synthase results in a decrease in mitochondrial membrane potential (mMP) and an increase in oxygen consumption. To provide a more integrated analysis of mitochondrial dynamics, this article describes the use of high-resolution fluorespirometry to measure the simultaneous response of oxygen consumption and mitochondrial membrane potential (mMP) to physiologically relevant concentrations of ADP. This technique uses tetramethylrhodamine methylester (TMRM) to measure mMP polarization in response to ADP titrations following maximal hyperpolarization with complex I and II substrates. This technique can be used to quantify how changes in health status, such as aging and metabolic disease, affect the sensitivity of mitochondrial response to energy demand in PBMCs, T-cells, and monocytes from human subjects.

Author Spotlight: New Insights into PBMC Mitochondrial Responses Using Fluorespirometry

Methods for studying mitochondrial bioenergetics under physiologically relevant substrate concentrations in immune cells are limited. We provide a detailed protocol that uses high-resolution fluorespirometry to assess changes in the response of the mitochondrial membrane potential to energy demand in human T-cells, monocytes, and peripheral mononuclear cells.

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these ...

Culturing of Activated T Cells from Human Peripheral Blood Mononuclear Cells

Source: M&#248;lgaard, K., et al. <a href="https://app.jove.com/v/62984">Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells.</a> J. Vis. Exp. (2021).
In this video, we describe a protocol for human primary T cell activation. The incubation of na&#239;ve T cells with magnetic beads coated with anti-CD3 and anti-CD28 antibodies, followed by interleukin-2 or interleukin-15 treatment, leads to the activation of na&#239;ve T cells and their differentiation and proliferation into effector or memory T cells.

Source: M&#248;lgaard, K., et al. Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells. J. Vis. Exp. 
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