Name: isolation and characterization of adult cardiac fibroblasts and myofibroblasts
Uploaded: 2020-03-12T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts at JoVE.com

作者要感谢伊沃·卡拉伊齐奇博士对βSMA-GFP小鼠的感谢。本出版物中报告的研究得到了国家卫生研究院国家普通医学研究所（NIH）的支持，编号为R01GM118300（S.S.），国家国家卫生研究院生物医学成像和生物工程研究所根据编号 R21EB019509 （P.P.Y.） 和美国心脏协会的科学家发展补助金，奖励编号 17SDG33630187 （S.S.）。流动细胞测量分析在VUMC流细胞测量共享资源处进行，该资源由范德比尔特英格拉姆癌症中心（P30 CA68485）和范德比尔特消化疾病研究中心（DK0058404）支持。

这项研究严格坚持《国家卫生研究院实验室动物护理和使用指南》中的建议。范德比尔特大学机构动物护理和使用委员会批准了该协议（协议号：M1600076-01）。
1. 心脏解剖
<ol><li>解决方案准备
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<li>KHB 缓冲器<ol>
<li>使用搅拌棒，在 900 mL DDI 水中缓慢溶解 9.4 克克雷布斯-亨塞莱特缓冲液 （KHB） 粉末。 注：如果搅拌速度过快或搅拌时间过长，缓冲器会沉淀。KH...

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T., Eghbali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+fibrillar+collagen+types+I+and+III+and+basement+membrane+type+IV+collagen+gene+expression+in+pressure+overloaded+rat+myocardium.">Regulation of fibrillar collagen types I and III and basement membrane type IV collagen gene expression in pressure overloaded rat myocardium.</a> Circulatory Research. 67 (4), 787-794 (1990).</li><li>Vasquez, C., Benamer, N., Morley, G. 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纤维细胞是一个异质细胞组，由不同的标记组识别。用于识别成纤维细胞的蛋白质标记物是二叶素域受体2（DDR2）、纤维素、维他丁、胶原蛋白I和III，以及Thy1 15、16、17、18、19、20。15,16,17,18,19,20维门汀已用于识别未受伤的静止心脏成纤维细胞，成?...

心脏成纤维细胞，中质源细胞，除了在平衡1期间维持心脏结构外，在维持心脏的电传导和机械力方面起着重要作用。损伤后，这些细胞被激活，膨胀，并产生细胞外基质（ECM）蛋白质2。许多临床前研究表明，成纤维细胞是维持受伤心脏3的结构完整性的关键细胞调节器，以及负责ECM蛋白无节制生产和沉积的主要作用细胞，导致硬性疤痕形成和心力衰竭4。纤维细胞是一组异质细胞，很难从亲纤维的不适应特性中剖析它们的恢复功能。最近，确定了心肌损伤后两种不同成纤维细胞亚型的功能异质性，表明可以隔离不同的纤维细胞亚型，并研究其在伤口愈合中的作用。
获得纯成纤维细胞种群对于界定其在修复和纤维化中的作用至关重要。然而，多重纤维细胞标记的存在，识别其他细胞类型，使得分离一个基本上纯成纤维细胞种群6具有挑战性。一些优雅的研究已经设计了巧妙的方法来分离心脏成纤维细胞从未受伤和受伤的心肌。最流行和成熟的方法来丰富成纤维细胞是通过选择性粘附后酶组织消化7。
此外，基于细胞表面抗原的成纤维细胞荧光激活细胞分拣（FACS）也已成功描述8。在这项研究中，在酶消化之后，从小鼠心脏对间皮细胞按系系阴性（林：Ter119+CD45+CD31+）和gp38阳性（gp38+）分类。−Gp38_ve细胞根据col1+1和其他间质标记的共同表达被确认为成纤维细胞。虽然大多数组织消化是在解剖出培养皿中的心室后完成的，但最近一项研究调查了左心室中直接注入针头酶以分离肌细胞和非肌细胞（包括成纤维细胞9）的研究。在这种情况下，纤维细胞通过选择性粘附分离。
该协议使用三种方法描述了成纤维细胞的分离和浓缩。第一种是一种已经确立的方法，即酶消化后有选择性地粘附成纤维细胞。第二种方法主要用于主要分离损伤引起的阿尔法平滑肌肉表达肌纤维细胞。第三种方法涉及血液细胞和内皮细胞酶消化的心脏细胞悬浮液的连续磁耗竭。耗竭后，纤维细胞/肌纤维细胞根据抗原MEFSK4的存在而使用磁珠进行分离。最近，MEFSK4被描述为一种抗原，存在于静止和活化成纤维细胞上，使其成为纤维细胞识别和隔离的合适标记。当然，这里描述的所有方法都有独特的局限性。因此，强烈建议通过流量分析、免疫染色和半定量实时PCR来检查分离细胞群的纯度。然而，这些方法可以扩大，并添加额外的标记，以排除其他污染人群之前，利用成纤维细胞和肌纤维细胞种群进行关键的实验。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-fungal (Amphotericin B-solubilized; Fungizone)</td>
			<td>Sigma Aldrich</td>
			<td>A9528</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serium Albumin (BSA)</td>
			<td>Sigma</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase blend (Liberase Blendzyme 3 TH)</td>
			<td>Roche Applied Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>DDI water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DI water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12 with L-Glutamine and HEPES</td>
			<td>Life technologies</td>
			<td>11330057</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I(20U/mL)</td>
			<td>BioRad</td>
			<td>7326828</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline (dPBS) without Ca2+ and Mg2+</td>
			<td>Gibco</td>
			<td>13190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FC Blocker (Purified anti-mouse CD16/CD32)</td>
			<td>Tonbo Biosciences</td>
			<td>70-0161</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Life technologies</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>10% goat serum</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS) with Ca2+ and Mg2+</td>
			<td>Corning</td>
			<td>21-023-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>1M HEPES</td>
			<td>Corning</td>
			<td>25-060-Ci</td>
			<td></td>
		</tr>
		<tr>
			<td>Krebs-Henseleit Buffer powder</td>
			<td>Sigma</td>
			<td>K3753</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycoplasma prophylactic (Plasmocin)</td>
			<td>Invivogen</td>
			<td>ant-mpp</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate-Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x Red Blood Cell Lysis Buffer</td>
			<td>Miltenyi</td>
			<td>130-094-183</td>
			<td></td>
		</tr>
		<tr>
			<td>Slow-fade Mounting Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>Sodium bicarbonate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#946;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Type 1 Rat Collagen</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Antibodies</td>
			<td></td>
			<td></td>
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		<tr>
			<td>7AAD (stock: 1 mg/mL solution in DMSO)</td>
			<td>Molecular Probes</td>
			<td>A1310</td>
			<td>dilution = 1:1000; RRID =</td>
		</tr>
		<tr>
			<td>CD45-APC</td>
			<td>BD Bioscience</td>
			<td>559864</td>
			<td>dilution = 1:200; RRID = AB_398672</td>
		</tr>
		<tr>
			<td>CD31-PE</td>
			<td>BD Bioscience</td>
			<td>553373</td>
			<td>dilution = 1:200; RRID = AB_394819</td>
		</tr>
		<tr>
			<td>CD31</td>
			<td>BD Biosciences</td>
			<td>553370</td>
			<td>dilution = 1:250; RRID = AB_394816</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>BD Biosciences</td>
			<td>553076</td>
			<td>dilution = 1:250; RRID = AB_394606</td>
		</tr>
		<tr>
			<td>COL 1&#945;1</td>
			<td>MD Bioproducts</td>
			<td>203002</td>
			<td>dilution = 1:1000; RRID =</td>
		</tr>
		<tr>
			<td>Ghost dye violet 510 (Formulation: 1 uL/test in DMSO)</td>
			<td>Tonbo Biosciences</td>
			<td>13-0870</td>
			<td>dilution = 1:1000; RRID =</td>
		</tr>
		<tr>
			<td>Goat anti-mouse Alexa Fluor 488</td>
			<td>Molecular Probes</td>
			<td>A11029</td>
			<td>dilution = 1:200; RRID = AB_138404</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit-Cy3</td>
			<td>Southern Biotech</td>
			<td>4050-02</td>
			<td>dilution = 1:200; RRID = AB_2795952</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit-FITC</td>
			<td>Jackson Immunoresearch Laboratories</td>
			<td>711-165-152</td>
			<td>dilution = 1:200; RRID = AB_2307443</td>
		</tr>
		<tr>
			<td>Goat anti-rat Alexa Fluor 488</td>
			<td>Molecular Probes</td>
			<td>A11006</td>
			<td>dilution = 1:200; RRID = AB_2534074</td>
		</tr>
		<tr>
			<td>Goat anti-rat Alexa Fluor 647</td>
			<td>Thermo-Fisher</td>
			<td>A21247</td>
			<td>dilution = 1:200; RRID = AB_141778</td>
		</tr>
		<tr>
			<td>Periostin</td>
			<td>Santa Cruz</td>
			<td>SC67233</td>
			<td>dilution = 1:100; RRID = AB_2166650</td>
		</tr>
		<tr>
			<td>Vimentin</td>
			<td>Sigma Aldrich</td>
			<td>V2258</td>
			<td>dilution = 1:200; RRID = AB_261856</td>
		</tr>
		<tr>
			<td>&#945;-smooth muscle actin (&#945;SMA)</td>
			<td>Sigma Aldrich</td>
			<td>A2547</td>
			<td>dilution = 1:1000; RRID = AB_476701</td>
		</tr>
		<tr>
			<td>Fibroblast specific protein 1 (FSP1)</td>
			<td>Millipore 07-2274</td>
			<td>07-2274</td>
			<td>dilution = 1:100; RRID = AB_10807552</td>
		</tr>
		<tr>
			<td>CD45 Magnetic Beads</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 Magnetic Beads</td>
			<td>Miltenyi Biotec</td>
			<td>130-087-418</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-feeder cells-APC (MEFSK4)</td>
			<td>Miltenyi Biotec</td>
			<td>130-102-900</td>
			<td>dilution = 1:100; RRID = AB_2660619</td>
		</tr>
		<tr>
			<td>anti-APC Beads</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-855</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG-APC</td>
			<td>Miltenyi Biotec</td>
			<td>130-103-034</td>
			<td>dilution = 1:100; RRID = AB_2661598</td>
		</tr>
		<tr>
			<td>Donkey anti-rat Alexa Fluor405</td>
			<td>Abcam</td>
			<td>ab175670</td>
			<td>dilution = 1:100</td>
		</tr>
		<tr>
			<td>anti-AN2/NG2</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-455</td>
			<td>dilution = 1:11; RRID = AB_2651235</td>
		</tr>
		<tr>
			<td>Other Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m Filter</td>
			<td>Thermo Scientific</td>
			<td>723-2520</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm2 Cell Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Pipet</td>
			<td>Fisherbrand</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m Cell Strainer</td>
			<td>Fisherbrand</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Pipet</td>
			<td>Fisherbrand</td>
			<td>13-678-11D</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical Tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Plate</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry Tubes</td>
			<td>Falcon</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rocker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Edge Blade</td>
			<td>PAL</td>
			<td>62-0177</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GFP-&#945;SMA Reporter Mice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Separator Magnetic Field</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Separation Column</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qaigen Rneasy Mini Kit</td>
			<td>Qaigen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Ambion RNAqueous Micro Total Isolation Kit</td>
			<td>Ambion</td>
			<td>AM1931</td>
			<td></td>
		</tr>
		<tr>
			<td>BioRad iScript cDNA Syntehsis Kit</td>
			<td>BioRad</td>
			<td>1708891</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well Plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30G Needle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3 Laser Flow Cytometry Machine (BD LSRFortessa)</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 Laser Flow Cytometry Machine (BD FACSAria III)</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Data Acquiring Software (BD FACSDiva Software v8.0a)</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Data Analysis Software (FlowJo Software)</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of adult cardiac fibroblasts and myofibroblasts

心脏纤维化对损伤的反应是伤口愈合的生理反应。已努力研究和靶向减轻纤维化的纤维细胞亚型。然而，由于缺乏普遍接受的成纤维细胞标记来识别静止和活化成纤维细胞，成纤维细胞的研究一直受到阻碍。纤维细胞是异质细胞群，难以分离和特征化。提出的协议描述了三种不同的方法来丰富纤维细胞和肌纤维细胞从未受伤和受伤的小鼠心脏。使用标准可靠的协议来分离成纤维细胞，可以研究它们在平衡和纤维化调制中的作用。

使用 βSMA-GFP 报告小鼠显示肌纤维细胞分离的流量浇注方案 未受伤的心脏在βSMA-GFP报告鼠模型中显示没有可检测的GFP+细胞;因此，它们被用来为补偿后GFP通道的背景信号建立一个门（图2）。*SMA=细胞根据在MI后10天从受伤左心室的GFP表达情况排序。一小部分内皮细胞（GFP+/CD31+） 细胞;SD = 3.8% = 0.0164;n = 5） 和造血性 （GFP+/CD45+ 细胞）;SD = 3.18% = 0.01...

Watch this Scientific Journal Video about Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts at JoVE.com

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

获得成纤维细胞的纯种群对于研究其在伤口修复和纤维化中的作用至关重要。这里描述的是一种详细的方法，从未受伤和受伤的小鼠心脏中分离成纤维细胞和肌纤维细胞，然后通过免疫荧光、RTPCR、荧光辅助细胞分拣和胶原蛋白凝胶收缩。

成人心脏纤维细胞和肌纤维细胞的分离与表征

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

该协议使我们能够以显著的纯度分离静态和损伤引起的活化成纤维细胞种群。这种技术的主要优点是它的灵活性即兴。对于流，以及基于珠子的分离，可以结合抗体对污染细胞，以排除他们从分离的目标细胞。此技术主要用于心脏成纤维细胞分离。然而，它可用于从其他组织分离成纤维细胞。演示这个程序的将是米玲梅尔泽，一个本科实习生和大卫比尔，一个研究助理，从我的实验室。要准备一个六井板，用于在解剖期间储存六颗心，将两毫升冷KHB放入每个井中，然后将板放在冰上。用70%乙醇喷洒身体。定向它，使腹侧面对实验者。固定附属物以防止干扰。在不刺穿肝脏的情况下，切开腹部皮肤和肌肉。垂直切向胸骨，小心地打开胸骨，而不会刺穿心脏。接下来，继续切开肋骨，露出心脏。使用钳子，轻轻地将心脏从胸部中抬起，切开任何附着在心脏外部的肺或多余的组织。分离左心室。对于每次解剖，将心室放在六孔板的单独井中。首先，使用钳子反复挤压和搅拌KHB中心室，以去除多余的血液。将心室转移到一个清洁、无菌的 10 厘米方形板中。接下来，使用单刃刀片，快速将心室切碎成小块。加入一毫升胶原酶消化鸡尾酒，继续切碎。当碎片足够小，可以用一毫升微移子转移时，将它们转移到50毫升的圆锥管中。用两毫升鸡尾酒洗盘子两次，然后将洗涤转移到管子上。孵育管30分钟，然后使用五毫升移液器重新暂停手稿中描述的细胞。初始再增后，孵育细胞15分钟，然后使用10毫升移液器再次重新暂停。然后，将一个 40 微米细胞过滤器放在新的 50 毫升锥形管上，然后用 1 到 2 毫升 KHB 润湿，使应变器保持素。在消化悬浮液中加入25毫升KHB，然后重新悬浮。通过过滤器过滤悬浮液，根据需要更换过滤器。在400倍g和4摄氏度下离心10分钟。取出上流液，使用每颗心脏5毫升缓冲液将颗粒重新在1x RBC解液缓冲液中。在室温下将悬浮液孵育两分钟后，在室温下以 400 次 g 离心悬浮液，在室温下将悬浮液离心机 10 分钟。取出上一液，然后将颗粒重新在一毫升KHB中。然后，在悬浮液中加入九毫升 KHB。通过 40 微米的底片电池滤株将悬浮液过滤到新的 50 毫升锥形管中。在室温下以 400 倍 g 离心锥形管 10 分钟。最后，去除上一除骨剂，将颗粒重新在一毫升成纤维细胞介质或PBS中。确定细胞号后，可以通过手稿中描述的三种不同方法从悬浮液中分离成纤维细胞。要开始通过差分电分离，通过在每个井中加入两毫升的成纤维细胞介质来准备一个六井板。旋转板以覆盖井底。如果小鼠心脏细胞悬浮在PBS中，则将离心机重新悬浮在每颗心脏一毫升的成纤维细胞介质中。在每个井中加入一毫升的细胞悬浮液，相当于一颗心脏。旋转板以均匀分布细胞。每井添加一到两毫升的成纤维细胞介质，总体积高达每井四毫升。在37摄氏度下孵育4小时。由于成纤维细胞将选择性地粘附在油井上，因此可以通过移除和丢弃介质来隔离它们。用两毫升PBS清洗剩余的附着细胞，然后每井加入2至4毫升无菌成纤维细胞介质。在37度孵育，直到汇合，每两到四天更换一次介质。为了开始对CD45阳性造血细胞进行磁性标记和分离，将小鼠心脏细胞的悬浮液以500倍g离心5分钟。取出上流液，将细胞颗粒重新在一毫升的平衡缓冲液中。使用血细胞计计算细胞。再次离心细胞后，在平衡缓冲液中重新填充细胞颗粒。加入CD45正磁珠，混合好。在4摄氏度下孵育至少15分钟。通过添加平衡缓冲液和在 4 摄氏度下以 500 倍 g 离心 10 分钟来清洗细胞。取出上清液，并使用血细胞计计算细胞。将颗粒重新在平衡缓冲液中。通过 40 微米过滤器进行悬浮液，以防止细胞聚合堵塞分离柱。接下来，在合适的分离器的磁场中放置分离柱，并平衡该柱与至少三毫升的 PBS。将悬浮液倒入柱中，并在 15 毫升锥形管中收集流经，该流经将包含未标记的细胞。用三毫升平衡缓冲液清洗柱，并将洗涤与流式处理相结合。从分离器上拆下柱，将柱放在 15 毫升圆锥形管上。要将标有 CD45 阳性的细胞进行回流，移液器将五毫升的平衡缓冲液插入柱中，然后将柱牢牢地插入。将水闸管和流管以 500 倍 g 离心。使用血细胞计计算细胞。要完成分离，请遵循CD31阳性内皮细胞和 MEFSK4 阳性成纤维细胞的手稿中的规程，这些协议与 CD45 阳性细胞的协议类似。在心肌梗死后从α-SMA-GFP小鼠心脏分离出的单细胞上进行荧光激活细胞分选。代表性的浇注方案演示 GFP 阳性细胞共同表达 CD31、CD45 或 AN2。在受伤的小鼠心脏中，一小部分内皮细胞和造血细胞表达GP。然而，GFP阳性/CD31-阴性/CD45-阴性细胞没有表达N2，一个周字节标记。GFP阳性细胞表示α-SMA，胶原蛋白一阿尔法一，周一汀和维门汀。未受伤的成纤维细胞通过选择性粘附分离表达维门汀，但未表现出α-SMA、周利汀或胶原蛋白的表达。未受伤和激活的成纤维细胞均表示 MEFSK4 抗原。两种未受伤的成纤维细胞，通过选择性粘附分离，和肌纤维细胞，从α-SMA-GFP小鼠分离和排序，证明有能力收缩胶原蛋白。在孵化前彻底切碎组织是重要的。在基于珠子的分离之前，通过 40 微米过滤器进行悬浮液以防止细胞聚集非常重要。使用这种方法净化的细胞可以使用与紫水晶特异性抗体相关的磁珠进一步纯化，以确保高水平的纯度。随着新的珠结合抗体的提供，以及记者转基因小鼠，人们可以利用这些技术来分离和研究不同的细胞群，而不仅仅是成纤维细胞。

Research

JoVE Journal

Biology

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

成人的心肌细胞的分离和遗传操作共聚焦成像

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

科研

生物学

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

分离，培养和成年小鼠Cardiomyoctyes的功能分析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

隔离成年小鼠心肌细胞的细胞信号传导和文化及<em&gt;在体外</em&gt;心肌肥厚

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

从小鼠和人类食管隔离肌纤维母细胞

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

从成年小鼠的分离，培养和窦房结肌细胞的功能分析方法

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

从成年大鼠心脏同时分离高质量心肌细胞，内皮细胞和成纤维细胞

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

骨骼肌成纤维细胞外泌体的分离和表征

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...