Name: an optimized protocol to analyze glycolysis and mitochondrial respiration in lymphocytes
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Description: Watch this Scientific Journal Video about An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes at JoVE.com

The authors thank Dr. Michael N. Sack (National Heart, Lung, and Blood Institute) for support and discussion, Ms. Ann Kim for optimizing the B cell isolation protocol, Dr. Joseph Brzostowski for his help with microscopy, and Ms. Mirna Pe&#241;a for maintaining the animals used. This study was supported by the Intramural Research Programs of the National Institutes of Health, National Institute of Allergy and Infectious Diseases, and National Heart, Lung, and Blood Institute.

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The authors declare that they have no competing financial interests. Open Access fees for this article were provided by Agilent Technologies.

我々が開発したプロトコルは、その後、解糖およびミトコンドリア呼吸性能の違いを評価するために種々の濃度で細胞外フラックス分析に使用した純粋な、生存リンパ球サブセットの効率的な分離を可能にしました。このプロトコルは、リンパ球のために特別に設計されており、このような低い基礎代謝活性、脆弱性、低周波数とこれらの細胞型に関連する特別な考慮事項に対処して、できないことは、アッセイプレートに付着されます。そのため、以前に公開されたプロトコル11,12に比べて、私たちの方法は、免疫学の分野で働く研究者のためのより便利で、より良い最適化されたガイドを提供しています。純粋な、生存細胞集団を得る最適なコンフルエンスで細胞をプレーティングし、各ウェル中のタンパク質濃度に外フラックスアッセイ測定を標準化するなど、このプロトコルにはいくつかの重要なステップがあります。 私播種した細胞のnitial数は両方の光学顕微鏡により可視化することができ、また、タンパク質濃度の測定を用いて定量します。細胞数及びタンパク質濃度の間の線形相関は、タンパク質濃度が実際に異なるウェル間の細胞数の変化の影響を標準化する方法として使用することができることを確認しました。 タンパク質濃度にECARとOCR結果を標準化することにより、我々は示された、より低い細胞コンフルエンスにもかかわらず、いくつかの5×10 2.5個の細胞/ウェルのデータの品質を損なうことなく、ほとんどのアッセイで使用することができるように。しかし、細胞タイプおよびアッセイ間で変動し使用することができる細胞の下限。例えば、わずか5×10 1.25ような細胞は、B細胞OCR測定のために使用することができるが、さらに2.5×10 5細胞/ウェルを、同じ細胞型の解糖系の性能を評価するのに十分ではなかったです。したがって、限り、細胞数は、制限要因ではないとしてオーバーでメッキ約5×10 5リンパ球に相当する90％のコンフルエンスで、/ウェル、好ましいです。異なるサイズ-、先に活性化され、部分的に分化などの細胞に使用リンパ球を-されたときにさらなる最適化が必要になることがあります。以前に初代培養リンパ球を使用した場合に加え、死亡または瀕死の細胞はまた、測定されたタンパク質レベルに寄与することができるので、細胞数の指標としてのタンパク質濃度の信頼性を減少させる別の処理条件との間の細胞生存率の変化、があるかもしれません。このような場合には、細胞外フラックスアッセイを行う前に、フローサイトメトリーにより生細胞を選別するために役に立つかもしれません。 新たに単離された、高度に実行可能なリンパ球集団を使用して、我々のアッセイでは、我々は両方のOCRとECAR測定のための信頼性の高い機能的なデータを得ました。全ての細胞型は、ミトコンドリアストレス試験において同様に挙動しながら、細胞型間の著しい差がありました解糖負荷試験で観察されました。例えば、ナイーブT細胞の解糖系の性能は、脾細胞またはB細胞と比較して低く、それは、オリゴマイシンの添加によって変化しませんでした。この観察は、我々のプロトコルの有効性を確認し、以前に発表された研究7,30に沿ったものです。 結論として、私たちの方法は、細胞外フラックスアナライザーを用いてリンパ球の代謝活性を試験する効率的かつ便利な方法を提供し、それは代謝活性化の際に、免疫細胞の変化、細胞の分化や原因を探求する免疫学的研究の広い範囲で有用であることができますこのような感染症、自己免疫および血液学的悪性腫瘍などの疾患の表現型に。

抗原に対する免疫応答は、免疫活性化と免疫抑制との間にしっかりと規制のバランスです。免疫抑制は、単にこれらのイベントを阻害し、したがって、不要な免疫応答1-6を防止するのに重要であるのに対し、免疫活性化は、刺激に応答して急速な細胞増殖および遊走、ならびにサイトカイン産生、抗体分泌の増加食作用を駆動します。最近の研究は、直接リンクは、免疫細胞の活性化状態及び様々な代謝経路7の活性の間に存在することを示しています。免疫細胞は、オンとオフのエネルギー産生経路を切り替えることにより、休止および活性化状態の間でシフトすることができます。また、別の免疫細胞タイプの活性化の間に、それらの増加したエネルギー需要を燃料に異なる代謝戦略を使用し得ることが観察されています。例えば、Tリンパ球の活性化はほぼ完全解糖状態8に細胞を指示しながら</su&gt; pは、活性化Bリンパ球は、解糖および酸化的リン酸化9,10のバランスを使用します。これらの研究は、細胞の代謝に対する免疫細胞活性化の影響を調査することの重要性を指摘します。 リアルタイムでは、酸素消費速度（OCR）および細胞外酸性化率（ECAR）の同時測定は、酸化的リン酸化及び解糖の指標として、経路11から13を製造するエネルギーの状態に対処するための一般的な戦略です。これを達成するために、そのようなシーホースXF 96のような細胞外フラックス分析器は、日常的に使用されます。このような機器は急速に細胞タイプで、または異なる刺激条件にOCRとECARの変化を比較することができます。これまでに、免疫細胞を含む様々な細胞型は、これらのデバイスを使用して研究されています。しかし、特に免疫細胞のために設計され最適化されたプロトコルは使用できません。 免疫細胞、特にリンパ球は、OTとは異なりますいくつかの重要な方法で彼女の細胞型。リンパ球は、ex vivoでの条件14-16で長期間生存していない脆弱な細胞です。これは、例えば、細胞外フラックス解析において使用されるような必須栄養素を欠く次善の増殖培地中で培養しても大きな問題です。マクロファージおよび多くの細胞株とは異なり、リンパ球は、プラスチック表面に付着しません。したがって、ストレスを発生させることなく、分析プレートに添付することが重要です。最後に、いくつかのリンパ球亜集団は非常にまれであってもよく、必要に応じ、最適な量でそれらを収穫することは17-19挑戦的であるかもしれません。 ここでは、具体的には、リンパ球のために開発された最適化されたプロトコルを提供します。 、我々はdifferenにその休止状態の解糖および酸化的リン酸化の特性を示す20ノードマウスの脾臓およびリンパ節から単離した脾臓細胞、Bリンパ球およびナイーブCD4 + Tリンパ球を使用してT細胞密度。データは、細胞数に直接比例した端アッセイ細胞溶解物のタンパク質濃度を測定することにより、各ウェルについての最初の細胞数の違いを説明するために正規化しました。私たちのプロトコルは、細胞外フラックスアッセイのための実行可能なリンパ球の迅速な単離のためのガイドラインを提供するだけでなく、それはまた、データの品質を損なうことなく、次善の細胞濃度での作業を可能にします。

<table><tbody><tr><td>PBS (pH 7.2)</td><td>Gibco-ThermoFisher</td><td>20012-050</td><td>To make MACS buffer</td></tr><tr><td>Bovine Serum Albumin BSA</td><td>Sigma-Aldrich</td><td>A3803</td><td>To make MACS buffer</td></tr><tr><td>0.5 M EDTA, pH 8</td><td>Quality Biological</td><td>10128-446</td><td>To make MACS buffer</td></tr><tr><td>autoMACS rinsing solution</td><td>Miltenyi Biotec</td><td>130-091-222</td><td>Instead of PBS + EDTA to make MS buffer. Also used in autoMACS Pro Separator.</td></tr><tr><td>RPMI-1640</td><td>Gibco-ThermoFisher</td><td>11875-093</td><td>Contains phenol red and L-glutamine</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco-ThermoFisher</td><td>10437-028</td><td>For heat-inactivation, thaw frozen stock bottle in a 37 &amp;deg;C water bath and then inactivate at 56 &amp;deg;C for 30 min. Aliquot heat-inactivated serum for storage (&lt;em&gt;e.g.&lt;/em&gt;, in 50 ml conical tubes) and freeze at -20 &amp;deg;C until needed.&amp;nbsp;</td></tr><tr><td>Penicillin-Streptomycin (Pen Strep)</td><td>Gibco-ThermoFisher</td><td>15140-122</td><td>Combine Pen Strep with L-Glut 1:1 (if making 500 ml media, make a total of 12 ml Pen Strep/L-Glut); keep aliquots at -20 &amp;deg;C until ready to make media.</td></tr><tr><td>L-Glutamine 200 mM (L-Glut)</td><td>Gibco-ThermoFisher</td><td>25030-081</td><td>Component of RF10 and stress test media</td></tr><tr><td>Sodium pyruvate 100 mM</td><td>Gibco-ThermoFisher</td><td>11360-070</td><td>Component of RF10 and stress test media</td></tr><tr><td>HEPES 1 M</td><td>Gibco-ThermoFisher</td><td>15630-080</td><td>Component of RF10</td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100x)</td><td>Gibco-ThermoFisher</td><td>11140-050</td><td>Component of RF10</td></tr><tr><td>2-Mercaptoethanol (55 mM)</td><td>Gibco-ThermoFisher</td><td>21985-023</td><td>Component of RF10</td></tr><tr><td>Falcon 70 &amp;mu;m cell strainer</td><td>Falcon-Fischer Scientific</td><td>87712</td><td>Used in cell isolation</td></tr><tr><td>Monoject 3 ml Syringe, Luer-Lock Tip</td><td>Covidien</td><td>8881513934</td><td>Used in cell isolation</td></tr><tr><td>Falcon 15 ml Conical Centrifuge Tubes&amp;nbsp;</td><td>Falcon-Fischer Scientific</td><td>14-959-53A</td><td>Used in cell isolation</td></tr><tr><td>Falcon 50 ml Conical Centrifuge Tubes&amp;nbsp;</td><td>Falcon-Fischer Scientific</td><td>14-432-22</td><td>Used in cell isolation</td></tr><tr><td>ACK Lysing Buffer</td><td>Lonza</td><td>10-548E</td><td>Used in cell isolation</td></tr><tr><td>CellTrics 30 &amp;mu;m cell filter, sterile, single-packed</td><td>Partec CellTrics</td><td>04-004-2326</td><td>Used in cell isolation</td></tr><tr><td>B Cell Isolation Kit, mouse</td><td>Miltenyi Biotec</td><td>130-090-862</td><td>Used in cell isolation</td></tr><tr><td>Na&amp;iuml;ve CD4&lt;sup&gt;+&lt;/sup&gt; T cell isolation kit, mouse</td><td>Miltenyi Biotec</td><td>130-104-453</td><td>Used in cell isolation</td></tr><tr><td>LS Column</td><td>Miltenyi Biotec</td><td>130-042-401</td><td>Used in cell isolation</td></tr><tr><td>MidiMACS separator</td><td>Miltenyi Biotec</td><td>130-042-302</td><td>Magnet for separation</td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td>Holder for magnets</td></tr><tr><td>autoMACS Rinsing Solution</td><td></td><td>130-091-222</td><td>Rinsing solution for autoMACS Pro Separator</td></tr><tr><td>autoMACS Pro Separator Instrument</td><td>Miltenyi Biotec</td><td>N/A</td><td></td></tr><tr><td>2-Deoxy-D-glucose (2-DG)</td><td>Sigma-Aldrich</td><td>D8375-5G</td><td></td></tr><tr><td>Cell-Tak</td><td>Corning</td><td>354240</td><td>Cell adhesive. Take care to note concentration, as each lot is different (package is 1 mg).</td></tr><tr><td>Oligomycin</td><td>Sigma-Aldrich</td><td>75351</td><td></td></tr><tr><td>2,4-Dinotrophenol (2,4-DNP)</td><td>Sigma-Aldrich</td><td>D198501</td><td></td></tr><tr><td>Antimycin A</td><td>Sigma-Aldrich</td><td>A8674</td><td></td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>Halt Protease Inhibitor Cocktail</td><td>ThermoFisher Scientific</td><td>78429</td><td>Supplied as 100x cocktail, combine with RIPA to form 1x solution for lysis.</td></tr><tr><td>RIPA Buffer</td><td>Boston BioProducts</td><td>BP-115</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>ThermoFisher Scientific</td><td>23225</td><td>Follow manufacturer&#039;s instructions</td></tr><tr><td>Seahorse XFe96 FluxPak</td><td>Seahorse Bioscience</td><td>101085-004</td><td>Includes assay plates, cartridges, loading guides for transferring compounds to the assay cartridge, and calibrant solution.</td></tr><tr><td>Seahorse XF Base Medium</td><td>Seahorse Bioscience</td><td>102353-100</td><td>Used to prepare stress test media</td></tr><tr><td>Seahorse XFe96 Extracellular Flux Analyzer</td><td>Seahorse Bioscience</td><td></td><td></td></tr><tr><td>Stericup Sterile Vacuum Filter Units, 0.22 &amp;mu;m</td><td>EMD Millipore-Fisher Scientific</td><td>SCGPU10RE</td><td>Used to sterile filter media.</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>487031</td><td>Dissolved to 0.1 M and used to dilute Cell-Tak.</td></tr></tbody></table>

an optimized protocol to analyze glycolysis and mitochondrial respiration in lymphocytes

リンパ球は、順番に急速な細胞増殖、遊走及び分化、およびサイトカイン産生をもたらす細胞内シグナル伝達経路を活性化することにより、種々の刺激に応答します。これらの事象の全てをしっかり細胞のエネルギー状態にリンクされ、したがって、エネルギー産生経路を研究することは、これらの細胞の全体的な機能に関する手がかりを与えることができます。細胞外フラックス分析器は、多くの細胞型における解糖およびミトコンドリア呼吸の性能を評価するために一般的に使用される装置です。このシステムは、いくつか公表された報告で免疫細胞を研究するために使用されてきた、まだ特にリンパ球用に最適化された包括的なプロトコルが欠けています。リンパ球は、ex vivoでの条件に乏しい生き残る脆弱な細胞です。しばしばリンパ球サブセットはまれであり、かつ低細胞数での作業は避けられません。したがって、これらの困難に対処する実験戦略が必要とされます。ここでは、プロトコルを提供しますそれは、リンパ組織から生存可能なリンパ球の迅速な単離を可能にし、細胞外フラックスアナライザーでそれらの代謝状態の分析のため。さらに、我々は、異なる細胞密度で、いくつかのリンパ球サブタイプの代謝活性を比較した実験の結果を提供します。これらの観察は、我々のプロトコルも低い細胞濃度で一貫性のある、よく標準化された結果を達成するために使用することができ、したがって、それは、免疫細胞における代謝イベントの特性に着目し、将来の研究において広範な用途を有し得ることを示唆しています。

Watch this Scientific Journal Video about An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes at JoVE.com

リンパ球の解糖とミトコンドリア呼吸を分析するための最適化されたプロトコル |免疫学と感染症 |ビデオ

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

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.

リンパ球における解糖およびミトコンドリア呼吸を分析するために最適化されたプロトコル

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

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29.5K ビュー.  National Institutes of Health. 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.

ビデオ: An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

造血幹細胞細胞代謝の解析

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a ...

発生生物学

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

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

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&#8217;s metabolism and phenotype.

リアルタイム細胞外フラックス分析を用いた偏光M1、M2の骨髄由来マクロファージの代謝特性評価

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

免疫・感染学

Measuring Yeast Mitochondrial Respiration While Maintaining Cellular Context

Mitochondrial Respiration Quantification in Yeast Whole Cells

Metabolism is mainly coordinated by cellular energy availability and environmental conditions. Assays for knowing how cells adapt energetic metabolism to different nutritional and environmental conditions give valuable information to elucidate molecular mechanisms. Oxidative phosphorylation is the primary source of ATP in most cells, and mitochondrial respiration activity is a key component of oxidative phosphorylation, maintaining mitochondrial membrane potential for ATP synthesis. Mitochondrial respiration is often studied in isolated mitochondria that are missing the cellular context. Here, we present a method for quantifying mitochondrial respiration in yeast-intact cells. This method applies to any yeast species, although it has been generally used for Saccharomyces cerevisiae cells. First, the yeast growth in specific conditions is tested. Then, cells are washed and resuspended in deionized water with a 1:1 ratio (w/v). Cells are then placed in an oximeter chamber with constant stirring, and a Clark electrode is used to quantify oxygen consumption. Some molecules are sequentially placed into the chamber and selected according to this effect on the electron transport chain or ATP synthesis. ATPase inhibitor oligomycin is first added to measure respiration coupled to ATP synthesis. Afterward, an uncoupler is used to measure the maximal respiratory capacity. Finally, a mix of electron transport chain inhibitors is added to discard oxygen consumption unrelated to mitochondrial respiration. Data are analyzed using a linear regression to obtain the slope, representing the oxygen consumption rate. The advantage of this method is that it is specific for yeast mitochondrial respiration, maintaining the cellular context. It is essential to highlight that inhibitors used in mitochondrial respiration quantification could vary between yeast species.

Mitochondrial respiration in yeast whole cells is a valuable indicator of cell bioenergetics. Here, we present a protocol to quantify this phenotype applicable to different yeast species.

Metabolism is mainly coordinated by cellular energy availability and environmental conditions. Assays for knowing how cells adapt energetic metabolism to ...

生化学

Analysis of Human Natural Killer Cell Metabolism

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to promote survival, cellular proliferation, production of cytokines such as interferon gamma (IFN&#947;) and/or cytotoxicity programs. NK cell activation by cytokine stimulation requires a substantial remodeling of metabolic pathways to support their bioenergetic and biosynthetic requirements. There is a large body of evidence that suggests that impaired NK cell metabolism is associated with a number of chronic diseases including obesity and cancer, which highlights the clinical importance of the availability of a method to determine NK cell metabolism. Here we describe the use of an extracellular flux analyzer, a platform that allows real-time measurements of glycolysis and mitochondrial oxygen consumption, as a tool to monitor changes in the energy metabolism of human NK cells. The method described here also allows for the monitoring of metabolic changes after stimulation of NK cells with cytokines such as IL-15, a system that is currently being investigated in a wide range of clinical trials.

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.

ヒトナチュラルキラー細胞代謝の解析

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to ...

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. Macrophages are commonly classified as M1-like (inflammatory) and M2-like (anti-inflammatory) subtypes. M1-like macrophages are pro-inflammatory macrophages that get activated by LPS and/or pro-inflammatory cytokines such as INF-&#947;, IL-12 &#38; IL-2. M1-like polarized macrophages are involved in various diseases by mediating the host&#39;s defense to a variety of bacteria and viruses. That is very important to study LPS induced M1-like macrophages and their metabolic states in inflammatory diseases. M2-like macrophages are considered anti-inflammatory macrophages, activated by anti-inflammatory cytokines and stimulators. Under the pro-inflammatory state, macrophages show increased glycolysis in glycolytic function. The glycolytic function has been actively investigated in the context of glycolysis, glycolytic capacity, glycolytic reserve, compensatory glycolysis, or non-glycolytic acidification using extracellular flux (XF) analyzers.
This paper demonstrates how to assess the glycolytic states in real-time with easy-to-follow steps when the bone marrow-derived macrophages (BMDMs) are respiring, consuming, and producing energy. Using specific inhibitors and activators of glycolysis in this protocol, we show how to obtain a systemic and complete view of glycolytic metabolic processes in the cells and provide more accurate and realistic results. To be able to measure multiple glycolytic phenotypes, we provide an easy, sensitive, DNA-based normalization method for polarization assessment of BMDMs. Culturing, activation/polarization and identification of the phenotype and metabolic state of the BMDMs are crucial techniques that can help to investigate many different types of diseases.
In this paper, we polarized the na&#239;ve M0 macrophages to M1-like and M2-like macrophages with LPS and IL4, respectively, and measured a comprehensive set of glycolytic parameters in BMDMs in real-time and longitudinally over time, using extracellular flux analysis and glycolytic activators and inhibitors.

This protocol provides detailed and comprehensive methods for the isolation, culture, polarization, and measurement of the glycolytic metabolic state of live bone marrow-derived macrophages (BMDMs). This paper provides step-by-step instructions with realistic visual illustrations for workflow and glycolytic assessment of BMDMs in real-time.

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. ...

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.

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.

サイトカイン分化型ヒト初代T細胞におけるミトコンドリア呼吸のリアルタイムモニタリング

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

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.

好中球のミトコンドリア生体エネルギープロファイルのリアルタイム測定

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.

培養細胞およびマウス組織から単離とミトコンドリアの機能解析

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

生物学

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

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, pro-inflammatory (i.e., M1-like) monocytes/macrophages display more reliance on anaerobic glycolysis and less reliance on mitochondrial oxidative phosphorylation, whereas anti-inflammatory (M2-like) macrophages display more reliance on glucose and fatty acid oxidation in the mitochondria. Here, we describe in-depth protocols for extracting macrophages from the two major monocyte/macrophage reservoirs in the body, the spleen and bone marrow, as well as injured tissues such as the heart following myocardial infarction.
Macrophages or monocytes are extracted by immunomagnetic sorting by using antibody-tagged microbeads, which easily bind to cells without compromising their phenotypes. The extracted cells are then cultured in 96-well plates, followed by extracellular flux analysis using a metabolic flux analyzer. Both glycolysis and mitochondrial oxidative phosphorylation can be measured simultaneously in small numbers of cells (as little as 2-3 &#215; 105 cells). This method can easily be performed in 1 day and produces reliable and repeatable results. Ultimately, these methods help to enhance our understanding of metabolic changes during immune and inflammatory responses to injury and disease, which could lead to the development of novel therapeutic targets for immunometabolic pathways.

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.

脾臓および心臓マクロファージおよび骨髄単球における ex vivo 代謝フラックスの解析

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, ...

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.