この作品は、全国多発性硬化症協会（TA AECへの3021 - A1 / T）とテキサス大学サンアントニオ校によってサポートされていました。

試薬の調製 CAがない場合10X HBSSの一部でパーコールの9部を混合することにより、株式等張パーコール（SIP）、脳あたり4 mlを、準備+ +およびMg + +。 15 mlのポリプロピレンコニカルチューブに分注し、脳当たりのCa + +およびMg + +、2 mlのなく1X HBSSで70％でSIPを準備します。 注：これは、パーコールは室温で使用されることをお勧めします、風邪を使用する場合、細胞が塊に傾向があると細胞分離が少なく効率的です。 組織の収集と均質化氷冷した1 × HBSSで左心室経由でマウスと浸み込ませるの麻酔。脳と脊髄を摘出し、すべてのマウスを屠殺されるまで、フェノールレッドを含まないRPMIで維持する。 RPMI 3mlを含む7または15ミリリットルダウンスホモジナイザーの場所の組織は、優しく、さらに組織を解離するために締め付けフィッティング乳棒（Bサイズ）に続く、緩い乳棒（サイズ）で細胞懸濁液を作る。 RPMIで7 mlに細胞懸濁液の体積を完了します。 勾配は、セットアップ最終的に30％のSIPを作るために細胞懸濁液に、SIPの3 mlを加え徐々に層の70％SIPの上に10 mLの細胞懸濁液。これはプロシージャの中で最も重要なステップであり、70％、30％溶液の混合回避重力モードで設定されたピペットエイドを使用してください。非常に明確なフラットラインは70％-30％の接合部で表示する必要があります。 500G 18℃で30分間遠心操作する相間が乱されないように遠心分離機が最小限、あるいはまったくブレーキで停止することを確認してください。 転送ピペットを用いて、軽くチューブの上部からごみの層を除去し、1X HBSS 8mlを含むきれいなコニカルチューブに70％-30％の相間の2.0〜3.0ミリリットルを収集する。相間を含むパーコールは、3倍程度希釈されていることを確認し、18で500Gで反転しcentrifugre 7分で数回混ぜる℃に懸濁します細胞染色緩衝液1mlでペレットとは、1.5 mlチューブに移すと4℃で10,000 G 1分間℃でマイクロ遠心機を使用して、1つより多くの時間を洗う抗体染色ヤギCD16/CD32を精製した50μlに再懸濁し、ペレットは、Fc結合部位をブロックするために細胞の染色バッファーで1:200に希釈した。 10分間氷の上にインキュベートする。血球計算板を用いて細胞を数える。 氷上で30分間抗体のカクテルミックスとインキュベート（50μl）を加えます。各抗体は、最初に最適な希釈を評価するために滴定する必要があります。レーザーと、特定のフローサイトメーターのフィルタ設定の機能に従って、選択した適切な蛍光色素の組み合わせを使用してください。 細胞の染色バッファ100〜200μlの細胞の染色バッファー、再懸濁し、ペレットに細胞を洗浄し、血球計算器で直ちに分析し、あるいは細胞は4℃のPBSで調製した2％パラホルムアルデヒド（PFA）に再懸濁し、一晩保存することができます° C 。 代表的な結果： グラデーションを回転した後、70％-30％の相間には定義されている白いハロを持っているはず、と素朴なマウスから回収された細胞の定量は、マウスにつき3〜5 × 10 5細胞（脳プラス脊髄）でなければなりません。炎症を起こした脳は中枢神経系の侮辱や疾患モデル（図1）によって炎症細胞数の範囲かもしれない。 <img src="/files/ftp_upload/2348/2348fig1.jpg" alt="図1" /> 図1ピーク疾患におけるナイーブおよびEAE -脳から単核細胞の単離。脳細胞の懸濁液は、不連続70％/ 30％パーコール勾配を介して分離した。染色した細胞を氷上で30分間抗CD45抗体が続くのFc -ブロックとインキュベートした。細胞は、細胞の染色バッファーでリンスし、そしてLSR - IIの2％PFA買収前に修正されました。 CD45の強度は、3つの異なった集団が可視化するY軸、に測定されます。 CD45 ハイテク浸潤細胞の免疫表現型のさらなる特徴は、追加の抗体を使用して実装し、続いてフローサイトメトリーによって分析されます。

<ol>
	<li>Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+direct+characterization+of+resident+microglial+cells+from+the+normal+and+inflamed+central+nervous+system.">Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system.</a> Proceedings of the National Academy of Sciences of the United States of America. 88, 7438-7442 (1991).</li><li>Ponomarev, E. D., Shriver, L. P., Maresz, K., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cell+activation+and+proliferation+precedes+the+onset+of+CNS+autoimmunity.">Microglial cell activation and proliferation precedes the onset of CNS autoimmunity.</a> J. Neurosci. Res. 81, 374-389 (2005).</li><li>Ford, A. L., Goodsall, A. L., Hickey, W. F., Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+adult+ramified+microglia+separated+from+other+central+nervous+system+macrophages+by+flow+cytometric+sorting.+Phenotypic+differences+defined+and+direct+ex+vivo+antigen+presentation+to+myelin+basic+protein-reactive+CD4++T+cells+compared.">Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared.</a> J Immunol. 154, 4309-4321 (1995).</li><li>Cardona, A. E., Huang, D., Sasse, M. E., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+microglial+cells+for+RNA+analysis+or+flow+cytometry.">Isolation of murine microglial cells for RNA analysis or flow cytometry.</a> Nat. Protoc. 1, 1947-1951 (2006).</li><li>Juedes, A. E., Ruddle, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+and+infiltrating+central+nervous+system+APCs+regulate+the+emergence+and+resolution+of+experimental+autoimmune+encephalomyelitis.">Resident and infiltrating central nervous system APCs regulate the emergence and resolution of experimental autoimmune encephalomyelitis.</a> J. Immunol. 166, 5168-5175 (2001).</li><li>Prinz, M., Priller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tickets+to+the+brain:+Role+of+CCR2+and+CX(3)CR1+in+myeloid+cell+entry+in+the+CNS.">Tickets to the brain: Role of CCR2 and CX(3)CR1 in myeloid cell entry in the CNS.</a> J Neuroimmunol. , (2010).</li><li>Mildner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+in+the+adult+brain+arise+from+Ly-6ChiCCR2++monocytes+only+under+defined+host+conditions.">Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions.</a> Nat. Neurosci. 10, 1544-1553 (2007).</li><li>Djukic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+monocytes+engraft+in+the+brain,+differentiate+into+microglia+and+contribute+to+the+pathology+following+meningitis+in+mice.">Circulating monocytes engraft in the brain, differentiate into microglia and contribute to the pathology following meningitis in mice.</a> Brain. 129, 2394-2403 (2006).</li></ol>

利害の衝突は宣言されません。

正常および炎症のマウス組織から中枢神経系の白血球の細胞表面マーカーの分析は、1月3日数十年にわたって利用されている。分離プロトコルは密度遠心4によるミクログリアと白血球の分離に基づいています。ここでは、不連続パーコール勾配を用いて、CNSの白血球を分離するの迅速かつ効果的な方法を説明します。細胞の単離の後、次のような - ではなく、CD45に限られた?...

組織の均質化とグラデーション<ul><li>パーコール（Amersham社） </li><li>カルシウムとマグネシウムのない10Xハンクス平衡塩溶液、（ギブコ） </li><li>フェノールレッドを含まないRPMI培地1640（Gibco社） </li><li> 7 - mlまたは15 mlのダウンスティッシュグラインダ（VWR） </li></ul>フローサイトメトリー<ul><li>細胞の染色バッファー（Biolegend） </li><li>精製ラット抗マウスCD16/CD32（マウスBD Fcをブロック）、BD Pharmingen社</li><li>血球計（フィッシャー） </li><li>抗マウスCD45（クローン30 - F11）、CD4（クローンRM4 - 5）、CD19（クローン6D5）、BioLegend </li><li> 10Xリン酸塩はカルシウムとマグネシウムなしで、緩衝生理食塩水（サーモサイエンティフィック） </li><li>パラホルムアルデヒド（シグマアルドリッチ） </li><li> LSR II（BD Biosciences社）を使用して実行フローサイトメトリー分析</li></ul>

isolation of brain and spinal cord mononuclear cells using percoll gradients

感染症、外傷、自己免疫疾患または神経変性における中枢神経系（CNS）に浸潤する免疫細胞の単離は、しばしばそれらの表現型とエフェクター機能を定義する必要があります。組織化学的アプローチは、浸潤細胞の位置を決定するために、病理関連したCNSを分析するための手段です。しかし、現場組織化学および免疫蛍光染色の技法は、特定の組織に免疫細胞のサブタイプを特徴付けるために一度に使用できる抗体の数によって制限されています。したがって、フローサイトメトリーによる免疫フェノタイピングと一緒に組織学的なアプローチは、完全にローカルのCNS浸潤の組成を特徴づけるために不可欠です。このプロトコルは、不連続パーコール勾配以上のCNSの細胞懸濁液の分離に基づいています。現在の記事は、効率的効果的にフローサイトメトリーにより、単一のサンプル中の様々な免疫細胞集団の同定に利用することができる脳や脊髄の組織から単核細胞を単離するために迅速なプロトコルを記述します。

Watch this Scientific Journal Video about Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients at JoVE.com

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

現在の記事は、効率的効果的にフローサイトメトリー分析に利用することができる脳や脊髄の組織から単核細胞を分離するために迅速なプロトコルを記述します。

パーコール勾配を用いて脳と脊髄単核細胞の単離

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

isolation-brain-spinal-cord-mononuclear-cells-using-percoll

Research

JoVE Journal

Immunology and Infection

40.5K ビュー.  University of Texas at San Antonio - UTSA. 現在の記事は、効率的効果的にフローサイトメトリー分析に利用することができる脳や脊髄の組織から単核細胞を分離するために迅速なプロトコルを記述します。

ビデオ: Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

フローサイトメトリーによって負傷した脊髄組織における免疫細胞の定量的評価：セルの精製方法のための新しい使用法

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

免疫・感染学

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

脳浸潤白血球の分離

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

ヒト胸腺からの骨髄樹状細胞と上皮細胞の単離

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

神経膠腫浸潤末梢血単核細胞の単離およびフローサイトメトリー分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

コラゲナーゼEを用いてマウスコロンからのラミナプロプリア単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

ヒト粘膜リンパ組織におけるEx Vivo自然免疫応答を研究する扁桃単核細胞の単核細胞の単離

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

マイクロバブルを用いたヒト末梢血単核細胞サンプルからの浮遊選鉱ベースのT細胞の単離、活性化、および増殖(英語)

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

ミトコンドリア生体エネルギー分析のためのPBMCコレクションと凍結保存

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

著者スポットライト:凍結保存後の末梢血単核細胞における生体エネルギーパラメータの保存 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

ヒト末梢血単核細胞の凍結保存と生体エネルギー評価

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...