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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著者らは、開示することは何もありません。

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modification of Eagles Medium</td>
			<td>Gibco</td>
			<td>12430-054</td>
			<td>with 4.5g/L D-glucose, L-glutamine, 25mM HEPES and phenol red&#160;</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-buffered Saline&#160;</td>
			<td>Gibco</td>
			<td>14190-144&#160;</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&#160;</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>10,000U/ml Penicillin; 10,000&#956;g/ml Streptomycin&#160;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>200mM</td>
		</tr>
		<tr>
			<td>G418 sulfate&#160;</td>
			<td>Omega Scientific Inc.</td>
			<td>GN-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td>from Streptomyces alboniger</td>
		</tr>
		<tr>
			<td>HyClone HyQTase non-mammalian trypsin alternative&#160;</td>
			<td>Thermo Scientific</td>
			<td>SV30030.01</td>
			<td>in DPBS with EDTA&#160;</td>
		</tr>
		<tr>
			<td>Phase contrast hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td>0.1mm 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>10ml vials</td>
		</tr>
		<tr>
			<td>0.6ml 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>5mg/ml solution</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride injection</td>
			<td>Fort Dodge</td>
			<td>NDC: 0409-2051</td>
			<td>100mg/ml solution</td>
		</tr>
		<tr>
			<td>Dexmedetomidine hydrochloride injection</td>
			<td>Zoetis</td>
			<td>NDC: 54771-2805</td>
			<td>0.5mg/ml solution</td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride injection</td>
			<td>Lloyd</td>
			<td>NDC: 61311-481</td>
			<td>100mg/ml solution</td>
		</tr>
		<tr>
			<td>Carprofen injection</td>
			<td>Pfizer</td>
			<td>NDC: 61106-8501</td>
			<td>50mg/ml solution</td>
		</tr>
		<tr>
			<td>Buprenorphine hydrochloride injection</td>
			<td>Reckitt Benckiser</td>
			<td>NDC: 12496-0757</td>
			<td>0.3mg/ml ampuls</td>
		</tr>
		<tr>
			<td>1ml tuberculin syringes&#160;</td>
			<td>Covidien</td>
			<td>8881501400</td>
			<td></td>
		</tr>
		<tr>
			<td>26G x &#189; (0.45mm x 13mm) syringe needles&#160;</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&#160;</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&#34; x 4&#34;, 4-ply&#160;</td>
		</tr>
		<tr>
			<td>1.7ml 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&#160;</td>
			<td>FST</td>
			<td>17000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Cordless precision power drill</td>
			<td>Dremel&#160;</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&#34; or 0.8 mm bit diameter</td>
		</tr>
		<tr>
			<td>Microliter syringe</td>
			<td>Hamilton</td>
			<td>75</td>
			<td>Hamilton Microliter&#174; Syringes 700 series; 5&#956;l volume</td>
		</tr>
		<tr>
			<td>Microliter syringe needles</td>
			<td>Hamilton</td>
			<td>7762-06</td>
			<td>33G, small hub RN NDL, 1.5 in, point style 3, 6/PK</td>
		</tr>
		<tr>
			<td>Ethilon 3-0 black monofilament nylon sutures&#160;</td>
			<td>Ethicon</td>
			<td>1663</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stir bar</td>
			<td>VWR</td>
			<td>74950-296</td>
			<td>7.9mm diameter x 50mm length (5/16&#34; diameter x 2&#34; length)</td>
		</tr>
		<tr>
			<td>1L 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&#160;</td>
			<td>Sagent</td>
			<td>NDC: 25021-400</td>
			<td>1,000U/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% O2 / 5% CO2)</td>
			<td>available from local vendor</td>
			<td>N/A</td>
			<td>A-type, large cylinder&#160;</td>
		</tr>
		<tr>
			<td>20G x 1 &#189;&#34; aluminum hub blunt needles</td>
			<td>Kendall</td>
			<td>8881202363</td>
			<td>0.9mm x 38.1mm</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&#160;</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Worthington Biochemical Corp.</td>
			<td>LS002007</td>
			<td>&#62;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. 115mm, base I.D. 85mm, 203ml volume&#160;</td>
		</tr>
		<tr>
			<td>Stainless steel single edged razor blades&#160;</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>7ml glass Dounce tissue grinder</td>
			<td>Kontes</td>
			<td>KT885300-0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll Density Centrifugation Media&#160;</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&#956;m sterile nylon mesh cell strainers&#160;</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&#160;</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)&#160;</td>
			<td>Biolegend</td>
			<td>101228</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 700-conjugated rat IgG2b, &#954; (clone: RTK4530) isotype control&#160;</td>
			<td>Biolegend</td>
			<td>400628</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG2a, &#954; (clone: eBM2a) isotype control&#160;</td>
			<td>eBioscience</td>
			<td>17-4724</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-conjugated rat IgG2b, &#954; (clone: eB149/10H5) isotype control&#160;</td>
			<td>eBioscience</td>
			<td>12-4031-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5-conjugated rat IgG2b, &#954; (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)&#160;</td>
			<td>Biolegend</td>
			<td>515403</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue-conjugated mouse IgG1, &#954; (Clone: MOPC-21) isotype control&#160;</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>15ml polypropylene centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>21-103</td>
			<td>conical bottom&#160;</td>
		</tr>
		<tr>
			<td>50ml polypropylene centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>21-108</td>
			<td>conical bottom</td>
		</tr>
		<tr>
			<td>12x75mm round-bottom polypropylene FACS tubes&#160;</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&#160;</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 ...

isolation-flow-cytometric-analysis-glioma-infiltrating-peripheral

Research

JoVE Journal

Immunology and Infection

15.6K 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 Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

マウス腹腔細胞の分離

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

免疫・感染学

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

過ヨウ素酸シッフ染色で末梢血単核細胞中のグリコーゲンの検出

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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 ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

ヒト全血中のナチュラルキラー細胞溶解活性のフローサイトメトリー分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

ヒト子宮頸管ガンマ・デルタT細胞の単離およびフローサイトメトリー分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

リンパ管内皮細胞のフローサイトのマウス肝臓の消化

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.

This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells.

単核食細胞の多パラメータフローサイトメトリック解析のためのマウス全体の眼の消化

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. ...

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 ...