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 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

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（<...

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

Research

JoVE Journal

Immunology and Infection

15.6K 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 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.

小鼠腹膜腔细胞的分离

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

科研

免疫与感染

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.

与碘酸雪夫染色检测糖原的外周血单个核细胞

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.

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 ...

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.

人全血中NK细胞杀伤活性的流式细胞仪分析

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.

人类宫颈的γδT细胞的分离和流式细胞仪分析

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.

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

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.

全鼠眼的消化，用于单核噬细胞的多参数流细胞学分析

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.

使用微泡从人外周血单核细胞样品中分离、活化和扩增基于浮选的 T 细胞

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

用于线粒体生物能量学分析的 PBMC 采集和低温储存

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.

作者聚焦：冷冻保存后外周血单核细胞中生物能量参数的保存 

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.

人外周血单核细胞的冷冻保存与生物能量评价

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