Name: isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells
Uploaded: 2015-11-28T15:00:00.000+00:00
Duration: 12 min 52 s
Description: Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells at JoVE.com

この作品は、神経疾患および脳卒中（NIH / NINDS）MGCにR01-NS074387、R01-NS057711とR21-NS091555を付与する/国立研究所国立衛生研究所によってサポートされていました。 NIH / NINDSはPRLにR01-NS061107、R01-NS076991、R01-NS082311とR21-NS084275を付与。リアのハッピーハート、ミシガン大学総合がんセンターからの助成金は、MGCとPRLに授与。脳神経外科、医学のミシガン学校の大学の学部; NIH助成2UL1-TR000433でサポートされている臨床的および保健研究、ミシガン研究所。 NIH / NCI（国立癌研究所）助成金T32-CA009676に​​よってサポートされているミシガン大学のがん生物学のトレーニンググラント。臨床およびNIH / NINDSグラントT32-NS007222によってサポートされる基本的な神経科学におけるミシガントレーニングの大学。およびNIH / NIGMS（一般医科学研究所）でサポートされているミシガン医学者研修プログラムの大学は、T32-GM007863を付与します。作者博士はカリンMuraszkoと脳神経外科から受信した学術的リーダーシップと支援に感謝再び。 M.ダールグレン、D. Tomford、と優れた行政支援のためのS.ナポリへ。優れた技術支援のためのM. Dzamanへ。そして、ツァイス3D走査型電子顕微鏡の購入に向けての暖かい支援に感謝フィルF.ジェンキンスへ。また、脳単核細胞の単離のための密度遠心メディア媒介戦略の修正版が描かれたハーバード・メディカル・スクールのKuchrooの研究室を認めます。

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このプロトコルは、初期のマウスの脳の腫瘍微小環境を浸透させてきたPBMCの単離およびフローサイトメトリー分析のための堅牢で再現性のある方法について説明します。神経膠腫細胞懸濁液を定位マウス脳の線条体に移植された実験者によって指定された濃度で生成されます。次いで、マウスを実験的設計によって指定された所定の時点で安楽死させ、それらの脳を採取し、蛍光標識一次抗...

神経膠腫は、中枢神経系（CNS）内に形質転換されたグリアから生じる神経上皮脳腫瘍のクラスです。すべての神経膠腫の、世界保健機関（WHO）グレードIVの神経膠腫、またはグリア芽腫（GBM）は、1最も一般的で致命的です。 GBMは、現在の標準のケアテモゾロミド2と放射線プラス付随およびアジュバント化学療法に続いて可能な限り腫瘍切除で構成さに非常に難治性です。これらの致命的な癌は5年3後の疾患の生存患者のわずか5％の初期診断時からの生存の唯一の15〜18ヶ月の悲惨な予後を運びます。 血液脳関門（BBB）の存在は、脳内の4細胞（APC）のプロフェッショナル抗原提示の欠如、および真正なリンパ管構造の以前に同定されていない存在は、免疫特権としてGBMの概念をもたらしました。しかし、今、多くの研究笙これらの脳癌は確かに、単球、マクロファージ、および骨髄由来抑制細胞（のMDSC）5を含む 、原点に主に骨髄ある末梢の免疫細胞の動員を生み出すことワットGBMはまた、6,7-プロ腫瘍形成になるための脳常駐ミクログリアの活性に影響を与えます。そのようなCD8 + T細胞8とCD56 +ナチュラルキラー細胞9などのリンパ系細胞はまた、腫瘍微小環境内に存在しているが、はるかに少ない数で、思ったことは、（、腫瘍関連マクロファージに神経膠腫由来因子によって扇動免疫抑制機能のためであるとするTAM）10。 CD4 + T細胞は、GBMにも存在するが、この集団の多くはまた、CD25とのFoxP3、免疫調節性T（TのREG）細胞11のメーカーを表しています。 GBMの全体的な免疫抑制状態は、免疫学的脱出し、腫瘍の進行12のプロモーションで最高潮に達します。 13-19を開発するために、脳腫瘍immunosuppressonのメカニズムを克服するために取り組んできました。この作品の集大成が今新たに診断されたGBM（：NCT01811992 ClinicalTrials.gov識別子）を持つ患者のために組み合わせた細胞傷害性および免疫刺激治療を評価するために設計された臨床試験につながっています。 私たちの最新作は、マウスGL26およびラットCNS-1 GBM細胞は、βガラクトシド結合レクチンガレクチン-1（GAL-1）20を大量に生産することにより、抗腫瘍NK細胞免疫監視をブロックすることを示しています。これは、shRNAを媒介遺伝子ノックダウンを用いた神経膠腫細胞におけるGAL-1の発現を抑制することにより実証された。 インビトロ experimenTSは、GAL-1欠損神経膠腫細胞は、培養中に正常に増殖していることを示した、まだ同系C57BL / 6JまたはRAG1に頭蓋内移植後すぐに急速な拒絶を受けた- / -マウスでは、このように、このフォームで、TまたはB細胞の独立性を確立します腫瘍拒絶。抗アシアロGM 1抗血清またはモノクローナルNK1.1抗体とNK細胞の免疫枯渇は、GAL-1欠損神経膠腫の拒絶反応におけるNK細胞の役割を確立し、頭蓋内GAL-1欠損神経膠腫の成長の完全な回復につながりました。現在、GR-1 + / CD11bのの免疫枯渇+骨髄細胞は、このように、NK媒介ギャルの補助で骨髄細胞のための不可欠な補助的な役割を明らかにし、NK細胞の存在にもかかわらず、GAL-1欠損神経膠腫の拒絶反応を予防するのに十分であることを示しています-1欠損腫瘍溶解（未発表データ）。この予想外の結果は、末梢血単核細胞（PBMC）の単離と解析のための包括的なプロトコルを開発するために私たちをリードしてきました私たちはより良いGAL-1欠損神経膠腫の拒絶反応を述免疫浸潤イベントを特徴づ​​けることができるようにすぐに頭蓋内移植後の脳の腫瘍の微小環境を浸透させます。 この方法は、GL26-Citを呼ばれる構成的に発現mCitrine蛍光タンパク質は、蛍光顕微鏡21による直接の腫瘍細胞の可視化を可能にするマウスGL26神経膠腫細胞を用いて、ここに示されています。これらの細胞は、定位的に同系のC57BL / 6Jマウスの脳に移植され、前のマウス安楽死に24、48、または72時間増殖させます。神経膠腫浸潤PBMCを、次に抗-CD45を用いて単離し、免疫標識され、-GR-1、-CD11bおよびグランザイムBの細胞内免疫標識と一緒-NK1.1細胞表面抗体（GZMB）。抗体のこの特定の組み合わせは、腫瘍浸潤のGr-1 + / +骨髄系細胞のCD11bおよびNK1.1 + NK細胞、細胞型、我々は、Bを持っているの同定を可能にしますEENは、GAL-1欠損腫瘍拒絶に関与します。 GAL-1欠損GL26-のCIT神経膠腫の免疫浸潤プロファイルは、GL26-CIT-gal1iとしてここでいう、その後非が含まれているGAL-1 GL26-CIT-NTと呼ばれるの正常なレベルで発現する神経膠腫のものと比較します対照shRNAのヘアピンをターゲットに。プロトコルは、同所同系のC57BL / 6Jマウスの線条体にこれらの細胞を生着する方法についての説明が続いて試験管内でどのように文化GL26-Citの神経膠腫細胞上の説明で始まり。その後、フローサイトメトリー分析のための神経膠腫浸潤PBMCの単離及び免疫標識に必要な手順を列挙するために進みます。プロトコルは、標準的なデータ分析とグラフ表示の説明を終わります。 デモでは、両方のGr-1 + / CD11bの+骨髄系細胞およびNK1.1 + NK細胞は優先的に48内GAL-1欠損脳の腫瘍微小環境内に蓄積することが明らかになりました腫瘍移植の時間、これらの腫瘍は急速に完全な腫瘍溶解約1週間後の腫瘍生着20を受ける理由を説明するのに役立ちます結果。この方法は、脳への免疫浸潤に関する時間データが要求されるインビボでの異なる実験計画の数に容易に適応可能です。単一の実験者は、分析すべき試料の数に応じて、約4-6時間で神経膠腫浸潤PBMCのフローサイトメトリー分析するために脳の収穫からプロトコルを実行することができます。この方法はまた、特に腫瘍微小環境によって誘導される免疫抑制表現型を識別するよう脳に浸潤するものと比較するために、腫瘍担持マウスでのPBMCの循環プロフィールを特徴づけることを目的とした実験と組み合わせることができます。これと同じような方法の適用は、脳の腫瘍微小環境への末梢免疫細胞の輸送に関与する因子の理解を容易にするはずです。

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Modification of Eagles Medium</td><td>Gibco</td><td>12430-054</td><td>with 4.5g/L D-glucose, L-glutamine, 25 mM HEPES and phenol red&amp;nbsp;</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-buffered Saline&amp;nbsp;</td><td>Gibco</td><td>14190-144&amp;nbsp;</td><td>without calcium chloride or Magnesium chloride</td></tr><tr><td>Fetal bovine serum</td><td>Omega Scientific Inc.</td><td>FB-11</td><td></td></tr><tr><td>Penicillin Streptomycin&amp;nbsp;</td><td>Gibco</td><td>15140-122</td><td>10,000 U/ml Penicillin; 10,000 &amp;mu;g/ml Streptomycin&amp;nbsp;</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030-081</td><td>200 mM</td></tr><tr><td>G418 sulfate&amp;nbsp;</td><td>Omega Scientific Inc.</td><td>GN-04</td><td></td></tr><tr><td>Puromycin dihydrochloride&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P8833</td><td>from Streptomyces alboniger</td></tr><tr><td>HyClone HyQTase non-mammalian trypsin alternative&amp;nbsp;</td><td>Thermo Scientific</td><td>SV30030.01</td><td>in DPBS with EDTA&amp;nbsp;</td></tr><tr><td>Phase contrast hemocytometer</td><td>Sigma-Aldrich</td><td>Z359629</td><td>0.1 mm deep</td></tr><tr><td>Trypan blue stain</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>Sterile 0.9% NaCl injection, USP</td><td>Hospira</td><td>NDC: 0409-4888</td><td>10 ml vials</td></tr><tr><td>0.6 ml conical polypropylenemi microtubes</td><td>Genesee Scientific</td><td>22-272</td><td></td></tr><tr><td>Atipamezole hydrochloride injection</td><td>Orion Pharma</td><td>NDC: 52483-6298</td><td>5 mg/ml solution</td></tr><tr><td>Ketamine hydrochloride injection</td><td>Fort Dodge</td><td>NDC: 0409-2051</td><td>100 mg/ml solution</td></tr><tr><td>Dexmedetomidine hydrochloride injection</td><td>Zoetis</td><td>NDC: 54771-2805</td><td>0.5 mg/ml solution</td></tr><tr><td>Xylazine hydrochloride injection</td><td>Lloyd</td><td>NDC: 61311-481</td><td>100 mg/ml solution</td></tr><tr><td>Carprofen injection</td><td>Pfizer</td><td>NDC: 61106-8501</td><td>50 mg/ml solution</td></tr><tr><td>Buprenorphine hydrochloride injection</td><td>Reckitt Benckiser</td><td>NDC: 12496-0757</td><td>0.3 mg/ml ampuls</td></tr><tr><td>1 ml tuberculin syringes&amp;nbsp;</td><td>Covidien</td><td>8881501400</td><td></td></tr><tr><td>26G x &amp;frac12; (0.45 mm x 13 mm) syringe needles&amp;nbsp;</td><td>BD</td><td>305111</td><td></td></tr><tr><td>Surgical clippers</td><td>3M</td><td>9661</td><td></td></tr><tr><td>Providone-iodine solution</td><td>Aplicare</td><td>82-217</td><td>NDC: 52380-1905</td></tr><tr><td>Sterile petrolatum ophthalmic ointment&amp;nbsp;</td><td>Dechra</td><td>NDC: 17033-211</td><td></td></tr><tr><td>70% isopropyl alcohol prep pads</td><td>Kendall</td><td>6818</td><td></td></tr><tr><td>Sterile gauze</td><td>Covidien</td><td>8044</td><td>non-woven, 4&quot; x 4&quot;, 4-ply&amp;nbsp;</td></tr><tr><td>1.7 ml conical polypropylene microtubes</td><td>Genesee Scientific</td><td>22-281</td><td></td></tr><tr><td>Mouse stereotactic frame</td><td>Stoelting</td><td>51730</td><td></td></tr><tr><td>Surgical lamp</td><td>Philips Burton</td><td>CS316W</td><td>Coolspot II Variable Spotlight</td></tr><tr><td>Curved dissecting forceps</td><td>Ted Pella Inc.</td><td>5431</td><td></td></tr><tr><td>Colibri retractors&amp;nbsp;</td><td>FST</td><td>17000-03</td><td></td></tr><tr><td>Cordless precision power drill</td><td>Dremel&amp;nbsp;</td><td>1100-01</td><td>Stylus model; 7.2-Volt Lithium-Ion with Docking Station</td></tr><tr><td>Engraving Cutter</td><td>Dremel</td><td>105</td><td>1/32&quot; or 0.8 mm bit diameter</td></tr><tr><td>Microliter syringe</td><td>Hamilton</td><td>75</td><td>Hamilton Microliter Syringes 700 series; 5 &amp;mu;l volume</td></tr><tr><td>Microliter syringe needles</td><td>Hamilton</td><td>7762-06</td><td>33 G, small hub RN NDL, 1.5 in, point style 3, 6/PK</td></tr><tr><td>Ethilon 3-0 black monofilament nylon sutures&amp;nbsp;</td><td>Ethicon</td><td>1663</td><td></td></tr><tr><td>Magnetic stir bar</td><td>VWR</td><td>74950-296</td><td>7.9 mm diameter x 50 mm length (5/16&quot; diameter x 2&quot; length)</td></tr><tr><td>1 L glass screw-cap storage bottle</td><td>Corning</td><td>1395</td><td>Type 1, Class A borosilicate glass</td></tr><tr><td>Heparin sodium&amp;nbsp;</td><td>Sagent</td><td>NDC: 25021-400</td><td>1,000 U/ml solution</td></tr><tr><td>Peristaltic pump</td><td>Cole Parmer Instruments Group</td><td>77200-60</td><td>Master Flex Easy Load II console drive</td></tr><tr><td>compressed carbogen (95% O&lt;sub&gt;2 &lt;/sub&gt;/ 5% CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>available from local vendor</td><td>N/A</td><td>A-type, large cylinder&amp;nbsp;</td></tr><tr><td>20 G x 1 &amp;frac12;&quot; aluminum hub blunt needles</td><td>Kendall</td><td>8881202363</td><td>0.9 mm x 38.1 mm</td></tr><tr><td>Polyurethane ice bucket</td><td>Fisherbrand</td><td>02-591-45</td><td>Capacity: 0.152 oz. (4.5L)</td></tr><tr><td>Collagenase (Type I-S)</td><td>Sigma Aldrich</td><td>C1639</td><td>from Clostridium histolyticum&amp;nbsp;</td></tr><tr><td>Deoxyribonuclease I</td><td>Worthington Biochemical Corp.</td><td>LS002007</td><td>&gt;2,000 Kunitz units per mg dry weight</td></tr><tr><td>Antistatic polystyrene hexagonal weighing dishes</td><td>Ted Pella Inc.</td><td>20157-3</td><td>top I.D. 115 mm, base I.D. 85 mm, 203 ml volume&amp;nbsp;</td></tr><tr><td>Stainless steel single edged razor blades&amp;nbsp;</td><td>Garvey</td><td>40475</td><td></td></tr><tr><td>Bone rongeurs</td><td>FST</td><td>16001-15</td><td></td></tr><tr><td>Hemostat</td><td>FST</td><td>13014-14</td><td></td></tr><tr><td>Large dissection scissors</td><td>Ted Pella Inc.</td><td>1316</td><td></td></tr><tr><td>Small dissection scissors</td><td>FST</td><td>14094-11</td><td></td></tr><tr><td>Blunt end forceps</td><td>Ted Pella Inc.</td><td>13250</td><td></td></tr><tr><td>7 ml glass Dounce tissue grinder</td><td>Kontes</td><td>KT885300-0007</td><td></td></tr><tr><td>Percoll Density Centrifugation Media&amp;nbsp;</td><td>GE Healthcare</td><td>GE17-0891-01</td><td></td></tr><tr><td>10 ml serological pipettes</td><td>Genesee Scientific</td><td>12-104</td><td>single use; sterile</td></tr><tr><td>70 &amp;mu;m sterile nylon mesh cell strainers&amp;nbsp;</td><td>Fisherbrand</td><td>22363548</td><td></td></tr><tr><td>Alexa Fluor 700-conjugated rat anti-mouse CD45 (clone: 30-F11)</td><td>Biolegend</td><td>103128</td><td></td></tr><tr><td>APC-conjugated mouse anti-mouse NK1.1 (clone: PK136)</td><td>eBiosciences&amp;nbsp;</td><td>17-5941-82</td><td></td></tr><tr><td>PE-conjugated rat anti-mouse Gr-1 (clone:RB6-8C5)</td><td>BD Pharmigen</td><td>553128</td><td></td></tr><tr><td>PerCP/Cy5.5-conjugated rat anti-mouse CD11b (clone: M1/70)&amp;nbsp;</td><td>Biolegend</td><td>101228</td><td></td></tr><tr><td>Alexa Fluor 700-conjugated rat IgG2b, &amp;kappa; (clone: RTK4530) isotype control&amp;nbsp;</td><td>Biolegend</td><td>400628</td><td></td></tr><tr><td>APC-conjugated mouse IgG2a, &amp;kappa; (clone: eBM2a) isotype control&amp;nbsp;</td><td>eBioscience</td><td>17-4724</td><td></td></tr><tr><td>PE-conjugated rat IgG2b, &amp;kappa; (clone: eB149/10H5) isotype control&amp;nbsp;</td><td>eBioscience</td><td>12-4031-82</td><td></td></tr><tr><td>PerCP/Cy5.5-conjugated rat IgG2b, &amp;kappa; (clone: RTK4530) isotype control</td><td>Biolegend</td><td>400632</td><td></td></tr><tr><td>Pacific Blue-conjugated mouse anti-mouse granzyme B (Clone: GB11)&amp;nbsp;</td><td>Biolegend</td><td>515403</td><td></td></tr><tr><td>Pacific Blue-conjugated mouse IgG1, &amp;kappa; (Clone: MOPC-21) isotype control&amp;nbsp;</td><td>Biolegend</td><td>400151</td><td></td></tr><tr><td>Cytofix/Cytoperm</td><td>BD</td><td>554714</td><td></td></tr><tr><td>15 ml polypropylene centrifuge tubes</td><td>Genesee Scientific</td><td>21-103</td><td>conical bottom&amp;nbsp;</td></tr><tr><td>50 ml polypropylene centrifuge tubes</td><td>Genesee Scientific</td><td>21-108</td><td>conical bottom</td></tr><tr><td>12 mm x 75 mm round-bottom polypropylene FACS tubes&amp;nbsp;</td><td>Fisherbrand</td><td>14-956-1B</td><td></td></tr><tr><td>FACSAria Special Order Research Product flow cytometer/cell sorter</td><td>BD&amp;nbsp;</td><td>650033</td><td></td></tr><tr><td>Class II Biological Safety Cabinet</td><td>The Baker Company</td><td>SG603</td><td>model: SterilGARD III Advance</td></tr></tbody></table>

isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells

私たちの研究室では最近、癌細胞はβガラクトシド結合レクチンガレクチンの発現が欠損しレンダリングされた場合に、ナチュラルキラー（NK）細胞はすぐに頭蓋内移植後に同所移植したマウスGL26およびラットCNS-1悪性神経膠腫を根絶することができることを実証しました-1（GAL-1）。より最近の研究は現在、GR-1 + / CD11bの+骨髄系細胞の集団がこの効果に重要であることを示しています。優れたNK細胞と骨髄細胞は、我々は、神経膠腫浸潤末梢血単核細胞（PBMC）の単離および分析のための総合的なプロトコルを開発したGAL-1欠損腫瘍拒絶を付与するために協力するメカニズムを理解するために。この方法は、shRNAのノックダウンを経由してGAL-1欠損レンダリングされたものとGAL-1を発現するGL26の神経膠腫の腫瘍微小環境へのPBMCの浸潤を比較することによって、ここに示されています。プロトコルはどのように文化の説明から始まり、GL26のCEを準備同系のC57BL / 6Jマウスの脳への接種のためにLLS。その後、初期の脳の腫瘍微小環境から神経膠腫浸潤PBMCの単離およびフローサイトメトリー解析に必要な手順について説明します。この方法は、脳への免疫浸潤に関する時間データが要求されるインビボでの実験計画の数に適応可能です。神経膠腫浸潤PBMCを、すぐに独立した実験を通して腫瘍一致した時点から観察同様の細胞数と24時間後に腫瘍生着などの頭蓋内腫瘍から単離することができるような方法は、高感度で再現性の高いです。単一の実験者は、分析されるサンプルの数に応じておよそ4-6時間で神経膠腫浸潤PBMCのフローサイトメトリー分析するために脳から採取方法を実行することができます。代替の神経膠腫モデルおよび/または細胞特異的な検出抗体は、他のいくつかのIMMUNの浸潤を評価するために実験者の裁量で使用することができます手順全体への変更を必要とせずに、目的の電子細胞タイプ。

CD11bの対FSC-W→CD45対数→GR-1対SSC-W→FSC-H対SSC-A→SSC-H対FSC-A→：次のゲーティング戦略は、典型的な実験のために使用されています数対NK1.1。ゲイツ氏はその後GZMB式（図5A）に基づいて層別化されたGR-1 + / CD11bの+骨髄系細胞およびNK1.1 + NK細胞上に置きました。 NK1.1 + NK細胞およびGR-1 + / CD11bのSSC-Aは、NK細胞の小さいリンパサイズおよび骨髄細...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells at JoVE.com

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

初期の脳の腫瘍微小環境を入力する免疫細胞の数および活性化状態に対する時間依存の定量的データをもたらす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のための簡単​​な方法は、ここに提示しました。

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 ...

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JoVE Journal

Immunology and Infection

15.7K ビュー.  University of Michigan. 初期の脳の腫瘍微小環境を入力する免疫細胞の数および活性化状態に対する時間依存の定量的データをもたらす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のための簡単​​な方法は、ここに提示しました。 

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

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

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 ...

免疫・感染学

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

カルボキシフルオレセインスクシンイミジルエステル（CFSE）を用いてヒト神経膠芽腫のスロー非分裂細胞の同定および単離

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

医学

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

ソート幹細胞アッセイおよびフローの原発性脳腫瘍組織の処理

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Functional Analysis of Tumor-Infiltrating Myeloid Cells by Flow Cytometry and Adoptive Transfer

The tumor-infiltrating myeloid cell compartment represents a heterogeneous population of broadly immunosuppressive cells that have been exploited by the tumor to support its growth. Their accumulation in tumor and secondary lymphoid tissue leads to the suppression of antitumor immune responses and is thus a target for therapeutic intervention. As it is known that the local cytokine milieu can dictate the functional programming of tumor-infiltrating myeloid cells, strategies have been devised to manipulate the tumor microenvironment (TME) to express a cytokine landscape more conducive to antitumor myeloid cell activity. To evaluate therapy-induced changes in tumor-infiltrating myeloid cells, this paper will outline the procedure to dissociate intradermal/subcutaneous tumor tissue from solid tumor-bearing mice in preparation for leukocyte recovery. Strategies for flow cytometric analysis will be provided to enable the identification of heterogeneous myeloid populations within isolated leukocytes and the characterization of unique myeloid phenotypes. Lastly, this paper will describe a means of purifying viable myeloid cells for functional assays and determining their therapeutic value in the context of adoptive transfer.

This protocol provides reliable methods of solid tumor dissociation and myeloid cell isolation in murine intradermal or subcutaneous tumor models. Flow cytometry allows for phenotypic characterization of heterogeneous myeloid populations within the tumor microenvironment and sorting will demonstrate their functionality in the context of adoptive transfer.

The tumor-infiltrating myeloid cell compartment represents a heterogeneous population of broadly immunosuppressive cells that have been exploited by the ...

がん研究

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

虚血性マウス脳からの免疫細胞の単離およびフローサイトメトリー分析

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

フローサイトメトリーによる中枢神経系からのミクログリアおよび単球由来マクロファージの解析

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within patient samples of glioblastoma, the deadliest and most invasive brain cancer. Not only are quantitative methods beneficial for accurately describing diseased tissues, they can also potentially contribute to more accurate prognosis, diagnosis, and the development of tissue-engineered systems and replacements. In glioblastoma, glial cells, such as microglia and astrocytes, have been independently correlated with poor prognosis based on pathologist grading. However, the state of these cells and other glial cell components has not been well-described quantitatively. This can be difficult due to the large processes that mark these glial cells. Furthermore, most histological analyses focus on the overall tissue sample or only within the bulk of the tumor, as opposed to delineating quantifications based on regions within the highly heterogeneous tissue. Here, we describe a method for identifying and quantitatively analyzing the populations of glial cells within the tumor bulk and adjacent regions of tumor resections from glioblastoma patients. We used chromogenic immunohistochemistry to identify the glial cell populations in patient tumor resections and ImageJ to analyze percent coverage of staining for each glial population. With these techniques we are able to better describe the glial cells throughout regions of the glioma tumor microenvironment.

This protocol was developed to quantitatively identify tumor microenvironment components in glioblastoma patient resections using chromogenic immunohistochemistry and ImageJ.

患者の膠芽細胞腫切除における細胞微小環境の定量的免疫組織化学

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

実験的自己免疫性脳脊髄炎における中枢神経系におけるリンパ球浸潤のフロー細胞量分析

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

<meta charset="utf8"></head><body>HA-Collagen Iハイドロゲルシステムによる膠芽腫浸潤の再現

A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are inaccessible to surgical resection and are resistant to existing chemotherapies. Tissue-level fluid movement, or interstitial fluid flow (IFF), regulates GSC invasion in a manner dependent on the tumor microenvironment (TME), highlighting the need for model systems that incorporate both IFF and the TME. We present an accessible method for replicating the invasive TME in glioblastoma: a hyaluronan-collagen I hydrogel composed of human GSCs, astrocytes, and microglia seeded in a tissue culture insert. Elevated IFF can be represented by applying a fluid pressure head to the hydrogel. Additionally, this model can be tuned to replicate inter- or intra-patient differences in cellular ratios, flow rates, or matrix stiffnesses. Invasion can be quantified, while gels can be harvested for a variety of outcomes, including GSC invasion, flow cytometry, protein or RNA extraction, or imaging.

<meta charset="utf8"></head><body>著者スポットライト:間質性流体の流れを介した神経膠芽腫の浸潤を研究するための患者情報に基づく3Dモデル

We present a method for replicating the glioma tumor microenvironment at the invasive front that incorporates interstitial fluid flow. This model is a hyaluronan-collagen I hydrogel in a tissue culture insert where a fluid pressure head can be applied. Invasion can be quantified, and cells can be isolated or lysed.

間質流を有する浸潤性神経膠腫微小環境の光重合性ヒアルロン酸コラーゲンモデル

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are ...

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

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パーコール勾配を用いて脳と脊髄単核細胞の単離

カルボキシフルオレセインスクシンイミジルエステル（CFSE）を用いてヒト神経膠芽腫のスロー非分裂細胞の同定および単離

ソート幹細胞アッセイおよびフローの原発性脳腫瘍組織の処理

Functional Analysis of Tumor-Infiltrating Myeloid Cells by Flow Cytometry and Adoptive Transfer

虚血性マウス脳からの免疫細胞の単離およびフローサイトメトリー分析

フローサイトメトリーによる中枢神経系からのミクログリアおよび単球由来マクロファージの解析

患者の膠芽細胞腫切除における細胞微小環境の定量的免疫組織化学

実験的自己免疫性脳脊髄炎における中枢神経系におけるリンパ球浸潤のフロー細胞量分析

間質流を有する浸潤性神経膠腫微小環境の光重合性ヒアルロン酸コラーゲンモデル

間質流を有する浸潤性神経膠腫微小環境の光重合性ヒアルロン酸コラーゲンモデル