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

这项工作是支持的美国国立卫生研究院神经疾病和中风（NIH / NINDS）授予R01-NS074387，R01-NS057711和R21-NS091555到的MGC /研究所; NIH / NINDS授予R01-NS061107，R01-NS076991，R01-NS082311和R21-NS084275到PRL;从利亚的快乐心助学金，密歇根大学综合癌症中心颁发给MGC和PRL大学;神经外科，医学密歇根大学的大学部;密歇根州的临床规范研究所与健康研究，由美国国立卫生研究院资助2UL1-TR000433支持;由NIH / NCI（国家癌症研究所）资助T32-CA009676支持的密歇根大学癌症生物学培训资助;密歇根培训的大学临床和基础神经科学由美国国立卫生研究院/ NINDS支持授予T32-NS007222;和密歇根大学医学院科学家培训项目的大学由美国国立卫生研究院/ NIGMS（通用医学科学研究所）的支持给予T32-GM007863。作者一再次感谢来自卡琳Muraszko博士和神经外科接收的学术领导和支持;到M.达尔格伦，D TomFord的，和S. Napolitan高超的行政支持;以M. Dzaman优秀的技术援助;和菲尔楼詹金斯向购买蔡司3D扫描电子显微镜的慷慨支持。我们也承认Kuchroo实验室在哈佛医学院从脑单个核细胞分离的密度离心媒体介导的战略的修改后的版本被吸引。

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作者什么都没有透露。

这个协议描述一种可再生的方法的PBMC的分离和流式细胞仪分析已渗入早期鼠脑肿瘤微环境。在由被立体定位植入到小鼠脑的纹状体的实验者指定的浓度产生神经胶质瘤细胞悬浮液。小鼠然后安乐死在由实验设计指定的预定的时间点和他们的大脑收获和加工以分离这些免疫标记与荧光标记的初级抗体的组合胶质瘤浸润的PBMC;免疫标记细胞，然后通过流式细胞术进行定量。虽然这里给出的示范着重于?...

胶质瘤是一类从转化胶质中枢神经系统（CNS）内所产生的神经上皮脑癌。在所有的胶质瘤，世界卫生组织（WHO）Ⅳ级神经胶质瘤，或胶质母细胞瘤（GBM），是最常见和致命的1。 GBM是高度耐火到当前标准的转交它由肿瘤切除术，以随后辐射加伴随和辅助化疗与替莫唑胺2尽可能。这些致命的癌症随身携带的只有15〜18个月的生存率从最初诊断时，只有5％的患者生存的疾病预后极差5 年 1之后。 的血脑屏障（BBB）的存在，缺乏专业的抗原呈递细胞的（的APC），和脑4内的以前未知的存在真正的淋巴结构导致基底膜免疫特权的概念。然而，大量的研究现在SHO瓦特这些脑癌确实产生外周免疫细胞主要是粒细胞中的起源，其中包括单核细胞，巨噬细胞和髓源抑制细胞（肌源性干细胞）5的募集。 GBM还影响脑驻留小胶质细胞的活性而成为亲致瘤6,7。淋巴样细胞例如CD8 + T细胞8和CD56 +自然杀伤细胞9也是在肿瘤微环境中存在的，但在少得多的数量，想到了一个事实是由于策动胶质瘤衍生的因子对肿瘤相关巨噬细胞的免疫抑制功能（噬细胞）10。 CD4 + T细胞也存在于GBM，但许多这类人群也表达CD25和FoxP3的，免疫调节性T（ 调节性T细胞）11机。 GBM的整体免疫抑制状态的高潮在促进免疫逃逸和肿瘤进展12。 13-19。这项工作的高潮已经导致了旨在评估组合的细胞毒性和免疫刺激治疗的患者初诊GBM（ClinicalTrials.gov标识符:NCT01811992）临床试验。 我们最近的工作表明，小鼠GL26和大鼠CNS-1 GBM细胞通过产生大量的β半乳糖苷结合凝集素半乳糖凝集素1（GAL-1）20块抗肿瘤NK细胞的免疫监视。这证明通过抑制加仑-1在神经胶质瘤细胞使用shRNA介导的基因敲除的表达。 体外 experimenTS表明加仑-1缺失神经胶质瘤细胞中培养正常增殖，但颅内植入进同源的C57BL / 6J或RAG1之后不久进行快速排斥- / -小鼠 ，从而建立T-或B细胞的独立性对这种形式的肿瘤排斥。 NK细胞免疫耗竭具有抗唾液酸GM 1抗血清或导致颅内GAL-1缺失的脑胶质瘤生长完全恢复单克隆抗体NK1.1，建立NK细胞GAL-1缺失的神经胶质瘤抑制的作用。我们现在表明的Gr-1 + / CD11b的+髓样细胞的免疫耗竭足以防止加仑-1缺失胶质瘤排斥尽管NK细胞的存在，从而揭示在NK介导的伽援用于骨髓细胞一个不可或缺的辅助作用的-1缺陷型肿瘤溶解（未公布的数据）。这一出人意料的结果使我们制定一个全面的协议，用于外周血单核细胞的分离和分析（外周血单个核细胞），其渗透到脑肿瘤微环境很快颅内植入后，让我们可以更好地表征，上游GAL-1缺失的胶质瘤抑制免疫渗透活动。 该方法是这里演示用鼠标GL26胶质瘤细胞组成性表达mCitrine荧光蛋白，叫GL26-CIT，它通过荧光显微镜21个州允许直接肿瘤细胞的可视化。这些细胞被立体定位植入进同源C57BL / 6J小鼠的脑和被允许之前小鼠安乐死生长24，48，或72小时。胶质瘤浸润的PBMC，然后分离和免疫标记使用抗-CD45，-GR-1，​​-CD11b和-NK1.1细胞表面的抗体与细胞内的免疫标记为颗粒酶B（GZMB）。这种特异性抗体的组合允许肿瘤浸润的Gr-1 + / CD11b的+髓样细胞的鉴定和NK1.1 + NK细胞，细胞类型，我们有bEEN牵连GAL-1缺失的肿瘤排斥。的GAL-1缺失GL26-CIT胶质瘤免疫浸润轮廓，这里称为GL26-CIT-gal1i，然后比较该胶质瘤表达加仑-1称为GL26-CIT-NT的正常水平，包含一非易失的针对对照shRNA的发夹。该协议开始于上GL26-CIT胶质瘤如何来培养细胞的说明在体外 ，随后是关于如何原位嫁接这些细胞进同源C57BL / 6J小鼠的纹状体的解释。然后，它前进到枚举涉及分离和胶质瘤浸润的PBMC用于流式细胞术分析的免疫标记的步骤。协议结尾标准数据分析和图形表示的解释。 该演示可以发现，GR-1 + / CD11b的+骨髓细胞和NK1.1 + NK细胞优先积累在48中的GAL-1缺失的脑肿瘤微环境中小时肿瘤植入，因此这有助于解释为什么这些肿瘤迅速进行完整的肿瘤溶解大约一周后的肿瘤植入20。该方法是很容易适应一些，其中对免疫渗入脑颞数据，需要在体内不同的实验设计。单个实验者可以从大脑收获执行协议流胶质瘤浸润的PBMC的流式细胞术分析在约4-6小时取决于待分析的样品的数目。该方法还可以用实验的目的是表征循环的PBMC在荷瘤小鼠对与那些渗入脑如此识别由肿瘤微环境特异性诱导的免疫抑制的表型比较的信息相结合。这和类似的方法应用应有助于更好地理解所涉及的外周免疫细胞贩运到大脑肿瘤微环境的因素。

<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

我们的实验室已最近表明，天然杀伤（NK）细胞能够颅内植入后CNS-1恶性胶质瘤很快消除原位移植小鼠GL26和大鼠如果癌细胞在它们的β半乳糖苷结合凝集素的galectin表达呈现缺陷的-1（加仑-1）。更近期的工作现在显示的Gr-1 + / CD11b的+髓样细胞群是这种效果的关键。为了更好地了解由NK细胞和骨髓细胞协作以赋予加仑-1缺失的肿瘤排斥我们已经开发了全面的协议用于分离和胶质瘤浸润外周血单核细胞（PBMC）的分析的机制。该方法是在这里证明了外周血单个核细胞浸润比较成的肿瘤微环境GAL-1表达GL26胶质瘤与那些通过shRNA的敲除呈现GAL-1缺失。该协议开始的描述如何培养并准备GL26 CELLS接种入同系的C57BL / 6J小鼠的大脑。然后，它解释了涉及从早期脑肿瘤微环境胶质瘤浸润的PBMC的分离和流式细胞术分析的步骤。该方法是适应若干体内 ，其中免疫渗入脑颞数据是必需的实验设计。该方法灵敏度高，重现性好，为神经胶质瘤浸润外周血单个核细胞可以从颅内肿瘤一旦被隔离24小时后移植瘤与时间点匹配的肿瘤遍布独立的实验中观察到类似的细胞计数。单个实验者可以从大脑收获执行该方法流胶质瘤浸润的PBMC的流式细胞术分析中大约4-6小时取决于待分析的样品的数目。替代胶质瘤模型和/或细胞特异性的检测抗体，也可以使用在实验者​​自行决定，以评估其它几个与免疫的渗透Ë细胞类型，而不需要改​​变对整体过程的兴趣。

下面浇注策略用于一个典型的实验:FSC-A对SSC-A→SSC-H与SSC-W→FSC-H与FSC-W→CD45与数→GR-1与CD11b的→ NK1.1与计数。盖茨放置的Gr-1 + / CD11b的+髓样细胞和NK1.1 + NK细胞，然后分层基于GZMB表达（图5A）。 Backgating那些鉴定为NK1.1 + NK细胞和Gr-1 + / CD11b的+髓样细胞在我们的实验上的FSC-A对SSC-A确认NK细胞的小尺寸淋巴和骨髓细胞的相对大尺寸（</s...

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

这里介绍的是胶质瘤浸润外周血单核细胞的分离和流式细胞术分析能够产生在进入早期脑肿瘤微环境的免疫细胞的数目和激活状态依赖于时间的定量数据一个直接的方法。

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

15.7K 次观看.  University of Michigan. 这里介绍的是胶质瘤浸润外周血单核细胞的分离和流式细胞术分析能够产生在进入早期脑肿瘤微环境的免疫细胞的数目和激活状态依赖于时间的定量数据一个直接的方法。 

视频: 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.

大脑和脊髓的单核细胞的分离，使用Percoll梯度

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

科研

免疫与感染

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.

慢分裂的细胞在人脑胶质瘤羧基荧光素琥珀酰亚胺酯（CFSE）的分离与鉴定

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.

加工的主要脑肿瘤组织的干细胞检测和流量排序

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 ...

癌症研究

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.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

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.

通过流式细胞术分析来自中枢神经系统的小胶质细胞和单核细胞衍生的巨噬细胞

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.

患者胶质母细胞瘤切除中细胞微环境的定量免疫组织化学

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.

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

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>使用透明质酸-胶原蛋白 I 水凝胶系统复制胶质母细胞瘤侵袭

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>作者聚焦：用于研究通过间质液流浸润胶质母细胞瘤的患者知情 3D 模型

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.

具有间质血流的侵袭性胶质瘤微环境的光聚合透明质酸-胶原蛋白模型

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 ...

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

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此视频中的章节

大脑和脊髓的单核细胞的分离，使用Percoll梯度

慢分裂的细胞在人脑胶质瘤羧基荧光素琥珀酰亚胺酯（CFSE）的分离与鉴定

加工的主要脑肿瘤组织的干细胞检测和流量排序

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

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

通过流式细胞术分析来自中枢神经系统的小胶质细胞和单核细胞衍生的巨噬细胞

患者胶质母细胞瘤切除中细胞微环境的定量免疫组织化学

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

具有间质血流的侵袭性胶质瘤微环境的光聚合透明质酸-胶原蛋白模型

具有间质血流的侵袭性胶质瘤微环境的光聚合透明质酸-胶原蛋白模型