Name: flow cytometry-based quantification and analysis of myocardial b-cells
Uploaded: 2022-08-17T14:50:00
Description: Watch this Scientific Journal Video about Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells at JoVE.com

这项研究由NHLBI授予Luigi Adamo的5K08HLO145108-03和1R01HL160716-01资助。
用于开展这项研究的Aurora流式细胞仪由NIH资助S10OD026859资助。我们感谢JHU Ross流式细胞术核心的支持。

本手稿中描述的所有实验均在约翰霍普金斯大学医学院IACUC的批准下进行。
1. 准备工作
<ol><li>准备FACS缓冲液，如 表1中所述。</li>
	<li>确保有足够的CO2 对动物实施安乐死。</li>
	<li>准备解剖空间（放置工作台垫并将胶带和解剖工具放在附近）。</li>
	<li>标记15 mL试管，在每个试管中放入3 mL含钙和镁的HBSS（参见材料表）并将它们放...

<ol>
	<li>Adamo, L., Rocha-Resende, C., Mann, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+role+of+B+lymphocytes+in+cardiovascular+disease.">The emerging role of B lymphocytes in cardiovascular disease.</a> Annual Review of Immunology. 38, 99-121 (2020).</li><li>Gowans, J. L., Knight, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+route+of+re-circulation+of+lymphocytes+in+the+rat.">The route of re-circulation of lymphocytes in the rat.</a> Proceedings of the Royal Society of London. Series B: Biological Sciences. 159 (975), 257-282 (1964).</li><li>Kunkel, E. J., Butcher, E. 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href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+myocardial+B+cells+mirror+changes+in+B+cells+associated+with+different+organs.">Developmental changes in myocardial B cells mirror changes in B cells associated with different organs.</a> JCI Insight. 5 (16), (2020).</li><li>Adamo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+B+cells+are+a+subset+of+circulating+lymphocytes+with+delayed+transit+through+the+heart.">Myocardial B cells are a subset of circulating lymphocytes with delayed transit through the heart.</a> JCI Insight. 5 (3), 139377 (2020).</li><li>Adamo, L., et al. <a target="_blank" 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越来越多的证据表明，B细胞在心肌生理学和心肌重塑/适应损伤的背景下起着重要作用7，8，9，10，11，12，13，36。流式细胞术是研究任何组织中免疫细胞群的绝佳工具，几乎是任何专注于心脏中B?...

B淋巴细胞是高度特化的免疫细胞，在适应性和先天免疫反应中都起着重要作用1。B细胞有两个主要群体：较小的B1细胞群，主要在胚胎生命期间产生，以及成年后在骨髓中产生的B2细胞群占主导地位1。在骨髓中成熟后，B细胞迁移到原代和次级淋巴器官。从那里，它们在通过血管和淋巴管的淋巴器官之间不断循环2。B细胞在其表面表达特异性抗体，其作为受体发挥作用。当B细胞遇到与其受体结合的抗原时，可以触发激活信号。活化的B细胞要么迁移到发现抗原的组织，要么回到骨髓，在那里它们可以成熟为产生抗体的浆细胞3，4。
最近，人们认识到心脏含有大量的B细胞。对啮齿动物的研究表明，B细胞在胚胎发育早期定植在心脏5，并且心肌相关B细胞大多是血管内幼稚的B2细胞粘附在内皮6，7上，其中B1细胞的比例很小7。仍有许多不确定的领域，但现有数据表明，B细胞在幼稚的心脏和心肌适应损伤的背景下都起着重要作用。
对幼稚鼠心脏的研究表明，在基线时心肌B细胞大多位于血管内间隙，粘附在内皮上（发现>95%的小鼠心脏B细胞位于血管内间隙）。发现这些B细胞的基因表达模式与从外周血分离的循环B细胞的基因表达模式不同。对B细胞缺陷动物的幼稚心脏和同系对照的分析发现，缺乏B细胞的动物心脏较小，射血分数较高6。所有这些证据表明，B细胞可能调节心肌生长和/或心肌功能，不仅间质，而且血管内B细胞都可能负责这种观察。还发现B细胞调节心肌常驻巨噬细胞的表型8。
一些研究表明，B细胞在心肌适应损伤的背景下起着重要作用8，9，10，11，12，13。B细胞在受伤的心脏中短暂积累，可能是通过CXCL13-CXCR5依赖机制11，13。从那里，B细胞通过几种机制促进不良心脏重塑，包括细胞因子介导的单核细胞募集9，12。此外，B细胞可以产生针对心脏蛋白的抗体，这些抗体可以通过几种机制促进心脏损伤的扩展和不良心脏重塑14，15，16，17，18，19，20，21，22，23，24，25.B细胞还可以通过分泌IL-10对受伤的心脏发挥保护作用10。
随着研究B细胞在幼稚和受伤心脏中的作用的小组数量的增加，定义共享协议以正确量化和评估心肌B细胞变得越来越重要，从而避免已经开始出现在文献中的不一致。事实上，据报道，B细胞都是啮齿动物心脏中最常见的免疫细胞之一7，并且其患病率明显低于骨髓细胞26，27，或者相当罕见28。同样，有几组人描述急性缺血性心肌损伤后心肌B细胞数量增加7、9、13，但有一组报告受损心肌B细胞数量没有变化29。对心脏免疫细胞的研究很少给出灌注条件的细节，对消化条件也没有达成共识。由于在啮齿动物心脏中，很大一部分B细胞在血管内，并且从心肌中提取免疫细胞高度依赖于所使用的消化方法，因此文献中报道的差异可能是器官灌注和组织消化差异的结果。
这里介绍的是一种基于流式细胞术的鼠心肌B细胞定量的详细方法，该方法通过优化灌注和消化条件来最大限度地提高B细胞回收率，并允许区分血管内与血管外心肌B细胞6。该协议是对区分血管内和间质免疫细胞的其他类似方案的改编和优化28，30，31。
在该协议中，我们将心肌灌注标准化，以消除漂浮在血管内空间中的B细胞，而无需去除粘附在微血管内皮上的生物学相关B细胞。此外，基于先前描述使用静脉注射抗体来区分血管内和间质免疫细胞的方案32，并利用B细胞表达表面标志物B22033的事实，我们演示了如何通过在动物处死和心脏灌注前立即血管内注射B220特异性抗体来区分血管内和血管外心肌B细胞。该协议与任何有兴趣包括幼稚和受伤心脏中心肌B细胞分析的科学家的研究相关。该协议的广泛实施将减少研究小组之间的不一致，将允许分析血管内和血管外心肌B细胞库的变化，从而促进心脏免疫学领域发现的进步。
总之，该协议代表了一种优化的工作流程，可通过流式细胞术量化和分析心肌B细胞，同时区分位于血管外空间和血管内间隙的细胞。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 700 anti-mouse/human CD11b Antibody</td>
			<td>101222</td>
			<td>BioLegend</td>
			<td>100 &#181;g 200 &#181;L</td>
		</tr>
		<tr>
			<td>(CellTreat 29481) Cell Strainer, 40 &#181;m, Blue</td>
			<td>QBIAP303</td>
			<td>Southern Labware</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL Natural Microcentrifuge Tube</td>
			<td>1605-0000</td>
			<td>SealRite, USA Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% Sodium Chloride Injection, USP</td>
			<td>114-055-101</td>
			<td>Quality Biological</td>
			<td>0.90%</td>
		</tr>
		<tr>
			<td>1.5 mL Natural Microcentrifuge Tube</td>
			<td>1615-5500</td>
			<td>SealRite, USA Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L Graduated TipOne Filter Tips</td>
			<td>11213810</td>
			<td>USA Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L Graduated TipOne Filter Tips</td>
			<td>11267810</td>
			<td>USA Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Centrifuge Tube, Plug Seal Cap, Polypropylene, RNase-/DNase-free</td>
			<td>430052</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Way Stop Valve, Polycarbonate</td>
			<td>SVPT951</td>
			<td>ECT Manufacturing</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2,2-Tribromoethanol</td>
			<td>T48402</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L Graduated TipOne Filter Tips</td>
			<td>11208810</td>
			<td>USA Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Way Stop Valve, Polycarbonate</td>
			<td>SVPT953</td>
			<td>ECT Manufacturing</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Polystyrene Round-Bottom Tube, 12 x 75 mm style</td>
			<td>352054</td>
			<td>Falcon, a Corning Brand</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube, Plug Seal Cap, Polypropylene, RNase-/DNase-free</td>
			<td>430290</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK (Ammonium-Chloride-Potassium) Lysing Buffer</td>
			<td>118-156-101</td>
			<td>Quality Biological</td>
			<td>Osmolality: 290 + or -5% mOsm/Kg H20</td>
		</tr>
		<tr>
			<td>Adapter 4x50ml, for 250 mL rectangular bucket in Rotor A-4-63</td>
			<td>5810759005</td>
			<td>Eppendorf</td>
			<td></td>
		</tr>
		<tr>
			<td>Adapter for 15 mL Centrifuge Tubes, 9 Tubes per Adapter, Conical Bottom for use with Rotor Model A-4-62</td>
			<td>22638289</td>
			<td>Eppendorf</td>
			<td></td>
		</tr>
		<tr>
			<td>Adapter for 15 round-bottom tubes 2.6 &#8211; 7 mL, for 250 mL rectangular bucket in Rotor A-4-62</td>
			<td>22638246</td>
			<td>Eppendorf</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum Foil 12 in x 75&#39; Roll .0007</td>
			<td>UPC 109153</td>
			<td>Reynolds Wrap</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia Induction Chamber - Mouse</td>
			<td>RWD-AICMV-100</td>
			<td>Conduct Science</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Luer Slip Tip Syringe with attached needle 25 G x 5/8 in., sterile, single use, 1 mL</td>
			<td>309626</td>
			<td>BD Becton, Dickinson and Company</td>
			<td></td>
		</tr>
		<tr>
			<td>Brandzig Ultra-Fine Insulin Syringes 29G 1cc 1/2&#34; 100-Pack</td>
			<td>CMD 2613</td>
			<td>Brandzig</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 421 anti-mouse CD19 Antibody</td>
			<td>115537</td>
			<td>BioLegend</td>
			<td>50 &#181;g/mL</td>
		</tr>
		<tr>
			<td>CAPS for Flow Tubes w/strainer mesh 35 &#181;m, Dual position for 12 x 75 mm tubes, sterile</td>
			<td>T9009</td>
			<td>Southern Labware</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Dioxide USP E CGA 940&#160;</td>
			<td>CD USPE</td>
			<td>AirGas USA</td>
			<td></td>
		</tr>
		<tr>
			<td>Cole-Parmer Essentials Low-Form Beaker, Glass, 500 mL</td>
			<td>UX-34502-46</td>
			<td>Cole-Parmer</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase 2</td>
			<td>LS004176</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>Connector brass chrome plated 1/4&#34; female NPT x 1/4&#34; barb</td>
			<td>Y992611-AG</td>
			<td>AirGas USA</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytek Aurora Flow Cytometer</td>
			<td></td>
			<td>Cytek Biosciences</td>
			<td></td>
		</tr>
		<tr>
			<td>Diss 1080 Nipple 1/4 BARB CP</td>
			<td>M-08-12</td>
			<td>AirGas USA</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I - 40,000 U</td>
			<td>D4527</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>Easypet&#160;3 - Electronic Pipette Controller</td>
			<td>4430000018</td>
			<td>Eppendorf</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic Balance, AX223/E</td>
			<td>30100606</td>
			<td>Ohaus Corp.</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5810R centrifuge</td>
			<td>5810R</td>
			<td>Eppendorf</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Research plus 1-channel variable pipettes</td>
			<td></td>
			<td>Eppendorf</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo 10.8.1</td>
			<td></td>
			<td>BD Becton, Dickinson and Company</td>
			<td></td>
		</tr>
		<tr>
			<td>GLACIERbrand, triple density Ice Pan (IPAN-3100)</td>
			<td>Z740287</td>
			<td>Heathrow Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (1x) - Ca2+ [+] Mg2+ [+]</td>
			<td>14025076</td>
			<td>gibco</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>H3506</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>Kelly Hemostats, Straight</td>
			<td>13018-14</td>
			<td>Fine Science Tools</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Slip Syringe sterile, single use, 20 mL</td>
			<td>302831</td>
			<td>BD Becton, Dickinson and Company</td>
			<td></td>
		</tr>
		<tr>
			<td>M1 Adj. Reg 0-100 PSI/CGA940</td>
			<td>M1-940-PG</td>
			<td>AirGas USA</td>
			<td></td>
		</tr>
		<tr>
			<td>McKesson Underpads, Moderate</td>
			<td>4033-CS150</td>
			<td>McKesson</td>
			<td></td>
		</tr>
		<tr>
			<td>Navigator Multi-Purpose Portable Balance</td>
			<td>NV2201</td>
			<td>Ohaus Corp.</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7.4 (1X) Ca2+ [-] Mg2+ [-]</td>
			<td>10010023</td>
			<td>gibco</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>PE anti-mouse/human CD45R/B220 Antibody</td>
			<td>103208</td>
			<td>BioLegend</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>PerCP/Cyanine5.5 anti-mouse CD45 Antibody</td>
			<td>103132</td>
			<td>BioLegend</td>
			<td>100 &#181;g 500 uL</td>
		</tr>
		<tr>
			<td>Petri dish, Stackable 35 mm x 10 mm Sterile Polystyrene</td>
			<td>FB0875711YZ</td>
			<td>Fisher Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Pkgd: Diss 1080 Nut/CO2/CO2-02</td>
			<td>M08-1</td>
			<td>AirGas USA</td>
			<td></td>
		</tr>
		<tr>
			<td>Powerful 6 Watt LED Dual Goose-Neck Illuminator</td>
			<td>LED-6W</td>
			<td>AmScope</td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlide Needle 25 G x 5/8 (0.5 mm x 16 mm)</td>
			<td>305122</td>
			<td>BD Becton, Dickinson and Company</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block) Clone 2.4G2 (RUO)</td>
			<td>553141</td>
			<td>BD Becton, Dickinson and Company Biosciences</td>
			<td>0.5 mg/mL</td>
		</tr>
		<tr>
			<td>R 4.1.1</td>
			<td></td>
			<td>The R Foundation</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor Blades</td>
			<td>9501250000</td>
			<td>Accutec Blades Inc</td>
			<td></td>
		</tr>
		<tr>
			<td>Regulator analytical two stage 0-25 psi delivery CGA320 3500 psi inlet</td>
			<td>Y12244A320-AG</td>
			<td>AirGas USA</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotor A-4-62, incl. 4 x 250 mL rectangular buckets</td>
			<td>Rotor A-4-62</td>
			<td>Eppendorf</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, plugged, 10 mL, sterile, non-pyrogenic/endotoxin-free, non-cytotoxic, 1 piece(s)/blister</td>
			<td>86.1254.001</td>
			<td>Sarstedt AG &#38; Co KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma label tape</td>
			<td>L8394</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectroFlo 3.0.0</td>
			<td></td>
			<td>Cytek Biosciences</td>
			<td></td>
		</tr>
		<tr>
			<td>Spex VapLock Luer Fitting, PP, Straight, Male Luer Lock x 1/8&#34; Hose Barb; 1/EA</td>
			<td>MTLL230-6005</td>
			<td>Spex</td>
			<td></td>
		</tr>
		<tr>
			<td>Std Wall Lab Tubing, Size S2, Excelon, 1/8&#34; ID x 3/16&#34; OD x 1/32&#34; Wall x 50&#39; Long</td>
			<td>CG-730-003</td>
			<td>Excelon Laboratory</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe PP/PE without needle, 3 mL</td>
			<td>Z683566</td>
			<td>Millipore Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>55-1199 (95-240)</td>
			<td>Harvard Apparatus</td>
			<td></td>
		</tr>
		<tr>
			<td>Thomas 3-Channel Alarm Timer TM10500</td>
			<td>9371W13</td>
			<td>Thomas Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube Rack, 12&#160;positions, 6 for 5.0&#160;mL and 15&#160;mL tubes and 6 for 25&#160;mL and 50&#160;mL tubes, polypropylene, numbered positions, autoclavable</td>
			<td>30119835</td>
			<td>Eppendorf</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube Rack, 12&#160;positions, for 5.0&#160;mL and 15&#160;mL tubes, polypropylene, numbered positions, autoclavable</td>
			<td>30119827</td>
			<td>Eppendorf</td>
			<td></td>
		</tr>
		<tr>
			<td>TYGON R-3603 Laboratory Tubing, I.D. &#215; O.D. 1/4 in. &#215; 3/8 in.</td>
			<td>T8913 (Millipore Sigma)</td>
			<td>Tygon, Saint-Gobain</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex-Genie 2</td>
			<td>SI-0236</td>
			<td>Scientific Industries, Inc.</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Dissecting Forceps with Guide Pin with Curved Tips</td>
			<td>89259-946</td>
			<td>Avantor, by VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Dissecting Scissors, Sharp Tip, 4&#189;&#34;</td>
			<td>82027-578</td>
			<td>Avantor, by VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Incubating Orbital Shaker, Model 3500I</td>
			<td>12620-946</td>
			<td>Avantor, by VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Zombie Aqua Fixable Viability Kit</td>
			<td>423102</td>
			<td>BioLegend</td>
			<td></td>
		</tr>
	</tbody>
</table>

flow cytometry-based quantification and analysis of myocardial b-cells

越来越多的证据表明，B淋巴细胞在心肌生理学和心肌适应损伤的背景下起着重要作用。然而，文献报告了关于心肌B细胞患病率的对比数据。据报道，B细胞既是啮齿动物心脏中最普遍的免疫细胞之一，要么存在，但患病率明显低于骨髓细胞，或者相当罕见。同样，几组人描述急性缺血性心肌损伤后心肌B细胞数量增加，但一组报告受损心肌B细胞数量没有变化。实施一种共享的、可重复的方法来评估心肌B细胞的患病率对于协调不同研究小组的观察结果至关重要，从而促进B细胞心肌相互作用研究的进展。根据我们的经验，文献中报道的看似相反的观察结果可能源于这样一个事实，即鼠心肌B细胞大多在血管内并与微血管内皮相连。因此，从小鼠心脏中回收的B细胞数量对用于清洁器官的灌注条件和所使用的消化方法非常敏感。在这里，我们报告了一个优化的协议，该协议以特定的方式考虑了这两个关键变量。该协议能够对鼠心肌B细胞的数量进行可重复的，基于流式细胞术的分析，并允许研究人员区分血管外与血管内心肌B细胞。

采集完成并收集所有事件后，应根据标准流式细胞术实践分析数据。分析的重点将根据每个实验的个别目标而有所不同。在这种情况下，进行血管内和血管外B细胞的定量，并将其表示为每毫克组织的细胞数。
使用光谱细胞仪时，建议从初始设门开始分析，以去除大小不在免疫细胞对应范围内的碎片，然后去除活死染色+信号。这允许检测对应于白细胞的CD45阳性细胞的清洁群?...

Watch this Scientific Journal Video about Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells at JoVE.com

Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells

在这里，我们报告了一种使用流式细胞术根据心肌B淋巴细胞在血管内或内皮空间中的位置对心肌B淋巴细胞进行定量和分化的协议。

基于流式细胞术的心肌B细胞定量和分析

A growing body of evidence shows that B-lymphocytes play an important role in the context of myocardial physiology and myocardial adaptation to injury. ...

文献报告了关于心肌B细胞患病率的对比数据。这可能源于灌注和消化技术的可变性。我们的方案能够对心肌B细胞进行可重复的流式细胞术分析。该技术最大限度地提高了B细胞的回收率，并减少了B细胞和其他心脏样本群体之间流式细胞术计数的变异性。这种优化的消化和分离方法可以很容易地修改，以实现扩展的抗体组合，以研究心脏中的不同免疫细胞类型。该方案也可以应用于其他器官，如肺或肝，以研究与这些器官相关的B细胞。首先，称一只 12 周大的雄性小鼠。用 100 微升 B220 抗体溶液加载 310-cc 胰岛素注射器。通过眶后注射注射稀释的B220抗体，并立即进行器官摘取和灌注。安乐死后，在上腹部用镊子握住小鼠的皮肤。用剪刀在皮肤上开一个两毫米的开口，用镊子剥开皮肤，露出筋膜层。在上腹部通过筋膜和腹膜切割。使用止血钳夹住剑突。在每侧锁骨中线水平处垂直切开胸壁，抬起前胸壁以显示纵隔内容物。使用镊子从后面牢固地握住主动脉。将注射器针头引入心脏顶点。以每分钟三毫升的速度灌注三毫升HBSS。切开主动脉并取出心脏。用HBSS在培养皿中清洗心脏。称量孤立的心脏并注意以毫克为单位的重量。用刀片在室温下将心脏切成小块。心形碎片的大小应约为 1.5 毫米。使用天平测量心脏组织的质量，并将60毫克切碎的心脏放入15毫升管中，用3毫升HBSS冰上消化。对未接受B220抗体注射的小鼠重复此过程，并将其放入装有3毫升HBSS标记的参比对照组织的管中。向每个含有碎组织的试管中添加酶。每毫克 DNase I 添加 0.5 微升，每毫克胶原酶 II 添加 0.5 微升，每毫克透明质酸酶添加 0.2 微升。将消化反应以每分钟300转的速度放入摇床中30分钟。将管子以小角度放置在振动器中，倾斜度约为25度。向每个 15 毫升反应管中加入 7 毫升 HBSS，并在 4 摄氏度下以 250 G 离心 5 分钟。倾析上清液后，将每个沉淀重悬于5毫升ACK裂解缓冲液中。在室温下孵育五分钟。向每个管中加入 10 毫升 PBS 后，混合并过滤到带有 50 微米过滤器的 40 毫升管中。用注射器柱塞粉碎过滤器上的任何碎屑，以确保所有材料都通过过滤器。通过过滤器添加PBS直到体积达到每颗心脏25毫升后，向参考对照组织管中额外添加25毫升PBS，并将体积分成两个不同的管。标记其中一个未染色的管子和另一个活的/死的。在4摄氏度下以250G离心5分钟后，倒出上清液并将未染色的管重悬于100微升PBS中，并将其他每个沉淀重悬于100微升活/死染色溶液中。在黑暗中在冰上孵育30分钟。向每个样品中加入500微升FACS缓冲液后，通过40微米过滤器将其转移到FACS管中。在4摄氏度下拒绝250G的FACS管五分钟并丢弃上清液后，将沉淀重悬于500微升FACS缓冲液中。向每个试管中加入50微升FC封闭溶液，未染色和活/死管除外。混合并孵育五分钟。向每个试管中加入50微升抗体混合溶液后，除未染色和活/死管外，在黑暗中孵育30分钟，用铝箔覆盖冰桶。与活/死污渍一样，用500微升FACS缓冲液再次洗涤，并重悬于300至500微升FACS缓冲液中。打开仪器控制软件。在窗口顶部，选择“获取”选项卡，然后单击“新建”。在库部分的“要过滤的类型”字段中，键入 PE、PerCP-Cy5.5、亮紫 421、Alexa Fluor 700 和 Zombie Aqua，选择氟-4 并在键入每个后单击添加。然后，单击下一步进入组选项卡。现在单击带有管子的符号以添加用于分析的心脏。单击引用组，将显示一个窗口。在荧光标签列中，选择 Zombie Aqua 作为荧光标签，通过单击每个标签旁边的垃圾红色图标删除任何其他标签。然后，在“控件类型”图标中，选择“单元格”。然后，单击保存。单击下一步。然后，单击标记选项卡。将显示一个表格。在每个氟-4列的第一行中键入相应的单元格标记，然后按Enter键。 单击“下一步”两次以转到“获取”选项卡。在实验样本的要记录的事件中，键入 1000 万，在参考组行的同一字段中键入 200， 000。单击保存并打开。在左下角，单击仪器控制。将电压设置为 FSC 50、SSC 175、SSC-B 175。选择采集控件。对于流速选项，请从下拉菜单中选择高。涡旋未染色的心脏管并将其牢固地放置在样品进样口上。确保绿色指示器箭头指向正确的样品。然后，单击记录并等待事件被记录。对活/死染色的心脏组织做同样的事情。选择“取消混合”菜单并设置控件。选中“自发荧光作为荧光标签”复选框。然后，单击下一步进入识别正/负总体选项卡。在此选项卡中，将显示前向散射区域与侧向散射区域的图。单击并拖动多边形的点，以在前向散点区域轴上选择 1 到 400 万之间的区域，在侧散点区域轴上选择 0.2 到 300 万之间的区域。在右侧的下一个图中，V5-A 将显示在 X 轴上。移动门以选择最右侧的一半峰值为正，将图左侧的区域选为负。在标题为“参考组 - 未染色”的图中，选择类似范围内的总体。对于未染色的，不得选择阳性/阴性。点击 现场解混 按钮。现在获取实验样品，涡旋管，并将其牢固地放置在SIP上。确保绿色指示器箭头指向正确的样品。然后，单击细胞仪软件的采集控制面板中的记录，并等待记录事件。选择“默认未混合工作表”选项卡。使用顶部的点图工具创建 FCS-A 与 SSC-A 图。使用面选择工具在 FSC-A 轴上选择大于 40 万的事件，在 SSC-A 轴上选择高于 80 万的事件。通过双击选区创建新图解，在此新图解中，右键单击 Y 轴标签并选择活/死染色。右键单击 X 轴标签并选择自发荧光 A.选择活/死轴上低于 10 到第五的所有事件。这些是活细胞。双击此选择。在新图中，选择 PerCP-Cy5.5A 作为 X 轴，选择 SSC-A 作为 Y 轴。选择PerCP-Cy5.5A右侧的种群。这些是免疫细胞。双击所选内容。在新图上，为 X 轴选择 FSC-A，为 Y 轴选择前向散射高度。选择遵循 FSC-A 值和 SSC-A 值相同的趋势的事件。这将选择单个细胞并消除双峰。双击此选择以在CD45阳性单细胞群中显示新图。从 CD45 阳性单细胞群图中，在 X 轴上选择明亮的紫色 421，在 Y 轴上选择 SSC-A。选择亮紫色 421 轴上右侧的人口。这是B细胞群。在此群体中双击两次以创建两个具有 B 细胞群的新图。设置第一个 B 细胞图，X 轴上有 PEA，Y 轴上有亮紫色 421A。右边的群体对应于血管间B细胞，左边的群体代表间质B细胞。同时选择两者。设置第二个B细胞图，X轴上为Alexa Fluor 700A，Y轴上为SSC-A。左边的群体对应于CD11b阴性细胞，右边的事件对应于CD11b阳性细胞。右键单击每个部分，然后单击门属性。单击“计数”和“父项”以显示事件数和父群体的百分比。记录事件数和百分比以进行进一步的数据分析。在去除幼稚的未受伤心脏中的双峰后，预计大约20%的CD45阳性细胞将是CD19阳性B细胞。在这些细胞中，平均有5.5%位于间质空间。94.5%在血管内间隙。在心脏中发现的整个B细胞群中，只有约1.6%对应于基于表面标志物CD11b的B1细胞，其他98.4%对应于B2细胞。当通过流式细胞术分析心肌B细胞时，按照方案正确灌注和切碎心脏至关重要。消化的心脏的适当过滤对于最大限度地恢复免疫细胞也至关重要。

Research

JoVE Journal

Immunology and Infection

Murine Model of CD40-activation of B cells

激活的B细胞CD40的小鼠模型

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

科研

免疫与感染

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

的分离，分化和人类的定量分泌抗体的B细胞从血液：酶联免疫斑点作为体液免疫的功能读出

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

流式细胞仪为基础的检测方法的NK细胞功能的监控

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

人外周血 NK 细胞活性评估流基于流式细胞仪检测细胞毒性检测

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

基于流式细胞仪的 Zika 病毒疗养血清测定登革热病毒的体外抗体依赖性

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

小鼠边缘区 b 细胞剪切流诱导迁移的体外分析

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...