我们要感谢吉森-马尔堡大学医院的临床团队的采血。这项工作由尤斯图斯·李比希大学资助。

本手稿中描述的所有采血、分离和分析方案均已由德国吉森大学机构审查委员会审查和批准。获得了患者同意将他们的样本纳入研究。分离和细胞培养的所有步骤均在生物安全柜下进行。
1. 静脉穿刺
<ol><li>准备采血所需的所有设备，包括消毒喷雾、无菌拭子、带 80 毫米管和多功能适配器的采血插管、止血带/血压袖带、Monovette 9 mL 肝素锂。 注意：EDTA作为抗凝剂也是有效的。</li>
	<li>从最合适的手臂静脉采集血液，通常是肘正中静脉或头静脉。</li>
	<li>使用止血带/血压袖带，轻压约 80 毫米/汞柱。</li>
	<li>用含有酒精的消毒喷雾对手套和穿刺部位进行消毒。让消毒的穿刺部位风干。</li>
	<li>静脉由于压力袖带的压力而突出。将针头（套管直径（外侧）21G / 0.8 mm，长度 19 mm）与静脉成 15°-20° 角，试图避免创伤并尽量减少探查。</li>
	<li>使用适当的系统采集血液，4 个装有 9 mL 血液的试管（一个实验者正确分离超过 7-8 个试管是有问题的）。</li>
	<li>采血后，将采集管置于黑暗中5分钟，以确保均匀抗凝。</li>
</ol>2. PBMC隔离
<ol><li>准备所有需要的解决方案，如下所述。</li>
	<li>将Dulbecco的平衡盐溶液（DPBS;浓度1x）和淋巴细胞分离培养基（1.077g/mL）置于室温（20-25°C）下。</li>
	<li>在室温下制备胎牛血清 （FBS），并将一个装有胎牛血清的无菌 50 mL 锥形管放在冰上。对于每个血样，需要 2 mL FBS。</li>
	<li>将冷冻容器储存在4°C，并将冷冻管预冷至4°C。</li>
	<li>将细胞培养基加热至37°C，培养基由RPMI 1640与50 mL FBS和青霉素50 U/mL链霉素50 U/mL组成。该溶液可在3°C下储存长达2个月。</li>
	<li>在无菌的 50 mL 锥形管中加入 8 mL DPBS。在无菌的 50 mL 锥形管中加入 15 mL 淋巴细胞分离培养基（培养基对光敏感，在开始分离前添加）。</li>
	<li>将 8 mL 血液加入 8 mL DPBS 中，并与 3 mL 塑料巴斯德移液管进一步小心混合。</li>
	<li>用 3 mL 塑料巴斯德移液管将血液/DPBS 混合物轻轻分层在淋巴细胞分离培养基上。要将第一层涂在介质上，请将管子倾斜 20°-30°，这将导致血液-PBS 混合物渗透到介质层中。</li>
	<li>将血液 - PBS混合物小心地铺在管的侧壁上，放在淋巴细胞分离培养基上。使用稳定的速度以保持血流恒定。</li>
	<li>在下一步中，将试管慢慢置于直立位置，将剩余的血液小心地分层在试管的侧壁上，放在血层上。</li>
	<li>在室温下以1000× g 离心10分钟，离心机带有带制动器的摆动桶转子。离心后，血液/PBMC混合物被分离成四层。顶层由血浆和血小板组成，第二层是PBMC层，其次是淋巴细胞分离培养基层，最后是底层的红细胞和粒细胞。不同的图层如 图 1 所示。</li>
	<li>用塑料巴斯德移液管去除2/3rd 的等离子层。</li>
	<li>使用 1 mL 移液管，在淋巴细胞分离培养基层上收集 PBMC，注意不要在样品中沾到任何培养基。</li>
	<li>将尖端放在 PBMC 层上方 1 毫米处。PBMC层不应被刺穿，否则介质将流过细胞。移液器的吸力将PBMC拉到该点，以便此时可以多次收集它们。 注意：为了最大限度地收集PBMC的数量，在程序结束时搜索表面上的其余部分，并尝试在那里收集细胞。为了稳定程序，可以将管子放置在表面上。</li>
	<li>将PBMC逐步转移到新的50 mL管中，直到层完全收获。加入 DPBS 至 25 mL 标记，洗去淋巴细胞分离培养基和其他残留物。</li>
	<li>在室温下以100× g 离心10分钟，并打开制动器。用真空泵或类似物除去上清液，注意不要损坏细胞沉淀。</li>
	<li>将沉淀重悬于 1 mL DPBS 中，并将 DPBS 加至 25 mL 标记。再次重复洗涤，然后重悬于适合后续步骤的培养基中。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65730/65730fig01.jpg"> 图 1：密度梯度离心示意图，用于说明不同层。 <a href="https://www.jove.com/files/ftp_upload/65730/65730fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
3. 冷冻保存
<ol><li>将冷冻容器冷却至4°C，在冰上冷却FBS。</li>
	<li>用 1 mL 移液管将 PBMC 沉淀重悬于 1 mL FBS 中，FBS 应在室温下。</li>
	<li>将DMSO与预冷的FBS在冰上1：5混合，最终浓度为20%DMSO，然后将FBS：DMSO混合物放回冰上。始终准备新鲜的溶液。</li>
	<li>将 FBS 中的 PBMC 细胞悬液转移到标记良好的 2 mL 冷冻管中。每个收集管使用一个冷冻管。在每 mL 1 x 107 和 5 x 107 之间调整细胞数。使用自动细胞计数仪测定细胞计数和活力。</li>
	<li>用 1 mL 移液管将 1 mL FBS：DMSO 混合物滴加至试管中，每秒约 1-2 滴。滴加可实现连续一致的混合。</li>
	<li>将试管放入预冷的冷冻容器中。将冷冻容器置于-80°C冰箱中24小时。冷冻容器提供每分钟-1°C的受控冷却。</li>
	<li>从-80°C冰箱中取出试管。从冰箱中取出后，将管子储存在液氮的气相中。记录每个样品的位置。</li>
</ol>4. 解冻
<ol><li>准备所有需要的溶液：含有 50 mL FBS 和青霉素 50 U/mL、链霉素 50 U/mL 的细胞培养基 RPMI 1640。该溶液可在 3°C 下储存长达 2 个月。</li>
	<li>将细胞培养基预热至37°C。 将 3 mL 预热细胞培养基加入无菌 50 mL 锥形管中。</li>
	<li>从液氮罐中取出样品。将样品在37°C的水浴中解冻约3.5分钟，一旦最后一块冰融化，立即从水浴中取出。管中应该仍然可以看到一块针头大小的冰块。 注意：DMSO对PBMC有害，尽快工作。</li>
	<li>用 1 mL 移液管从冷冻管中取出 cell：FBS：DMSO 混合物。将PBMC样品与制备的50 mL管中的细胞培养基混合。用 5 mL 培养基分 2 mL、2 mL 和 1 mL 三个步骤洗涤试管。</li>
	<li>将培养基转移到试管中。这样做是为了转移可能的细胞残基。在室温下以100× g 离心10分钟。</li>
	<li>弃去上清液，加入 1 mL 适合计划使用的培养基。细胞已准备好进行后续实验。 注意：然而，对于新鲜或冷冻细胞的功能测试，通常建议在基于淋巴细胞分离培养基的分离或细胞解冻后在培养箱中静置一段时间（通常过夜）。</li>
</ol>5. 细胞培养
<ol><li>分离或解冻后，在37°C的5%CO2 / 95%空气中培养细胞过夜。</li>
	<li>将细胞重悬于补充有 10% FBS、青霉素 50 U/mL、链霉素 50 U/mL 的 1 mL RPMI 培养基中。为了进一步使用，有许多可能性，根据测定的需要处理细胞。</li>
	<li>对于一般储存，使用无菌 6 孔细胞培养板，并在静置期后收获细胞。用 1 mL 移液管将 1 mL 细胞悬液转移到孔中，并加入 4 mL 细胞培养基。 注意：分离的PBMC的量因个体而异，每分离8mL血液，一个装有5mL细胞培养基的孔就足够了。然而，当从血沉棕黄层中分离PBMC时，PBMC的数量明显高于全血样品，因此应将细胞分成几个孔。</li>
	<li>让细胞在37°C下补充有5%CO2 的湿润气氛中静置24小时。 该孵育适用于新鲜分离的细胞以及冷冻保存的细胞。</li>
</ol>6. ATP测定
<ol><li>将解冻的PBMC重悬于补充有10%FBS，青霉素50U / mL，链霉素50U / mL的1mL RPMI培养基中。</li>
	<li>取样并确定细胞计数，然后用台盼蓝进行活死鉴别。从重悬细胞中取出 10 μL，并与 90 μL 细胞培养基混合。然后取 10 μL 并与 10 μL 台盼蓝混合。将细胞放入细胞计数室或自动细胞计数器中，并确定活/死细胞的数量。</li>
	<li>在 96 孔白色聚苯乙烯板中培养 100 μL 密度为 1 x 105 个细胞/100 μL 的细胞。</li>
	<li>让细胞在37°C下补充有5%CO2 的湿润气氛中静置24小时。</li>
	<li>用ATP测定法测定ATP浓度。<ol><li>使用ATP与荧光素结合时发生的光发射。发射的光可以用酶标仪进行评估。取出培养箱的板，冷却至室温15分钟。裂解细胞并放置5分钟。然后将监测试剂涂在细胞上，并根据制造商的说明进行测量。内标用于确定 ATP 水平。</li>
	</ol></li>
</ol>7. 高分辨率呼吸测量法
<ol><li>打开高分辨率氧谱仪，让它预热 30 分钟。</li>
	<li>根据 图1中描述的方案处理细胞。如 表1所示，准备所需的所有库存。</li>
	<li>将2.1mL呼吸缓冲液（表1）移液到两个高分辨率氧谱仪室中，并在37°C下使用腔室中的磁力搅拌棒（750rpm）连续搅拌缓冲液30分钟，直到获得极谱氧传感器的稳定氧通量信号。 注意：在氧谱仪的腔室中，借助极谱氧电极测量实时耗氧量（通量）和腔室的氧饱和度。必须执行背景校准以避免背景噪声并确保可靠的结果。</li>
	<li>根据制造商的协议对极谱氧传感器进行空气校准23.</li>
	<li>将分离的PBMC重悬于1mL线粒体呼吸培养基（MIR05，组成如 表1所示）中，并稀释至8×106 个细胞/ mL。</li>
	<li>空气校准后，从氧谱仪室中吸出呼吸介质，并在呼吸计的每个外倾角中加入 2.1 mL 细胞悬浮液。如果在测量过程中需要对腔室进行再氧合（参见 图 2 中 h 点的打开），腔室的氧饱和度不应低于 100 μM。</li>
	<li>通过插入塞子关闭腔室，腔室设计为可容纳 2.0 mL 体积。吸出新兴细胞悬液。</li>
	<li>在37°C下用位于腔室中的磁力搅拌器（750rpm）连续混合细胞悬浮液。等待约20分钟，直到获得稳定的信号。确定内源性呼吸（ 图2中的（a））。</li>
	<li>为了确定呼吸链的不同复杂活性，通过塞子的钛注射口注射用于线粒体呼吸的底物和抑制剂。在腔室中使用以下最终浓度。</li>
	<li>为了破坏细胞膜，通过腔室塞子的钛进样口加入 5 μL 8.1 mM 洋地黄皂苷，以去除 图 2 中的幼稚底物 （b），同时线粒体膜保持完整。</li>
	<li>通过腔室塞子的进样口加入底物2M谷氨酸和800mM苹果酸，并记录呼吸，直到获得稳定的信号。信号在2-4分钟后稳定下来。 注意：也可以使用丙酮酸等其他基材。</li>
	<li>通过腔室塞子的进样口加入 8 μL 500 mM 二磷酸腺苷 （ADP） 并记录呼吸，直到获得稳定的信号。 信号在图2中2-4分钟（d）后稳定下来。</li>
	<li>通过腔室塞子的进样口加入 20 μL 1 M 琥珀酸盐并记录呼吸，直到获得稳定的信号。 信号在图2中2-4分钟（e）后稳定下来。</li>
	<li>在0.5μL下逐步滴定1M羰基氰化物对三氟甲氧基苯腙（FCCP），直至不再增加。等待 2-4 分钟，直到信号稳定 （f） 如 图 2 所示。当呼吸没有进一步增加时，继续下一步。 注意：处理 FCCP 时要小心，因为它对人类有健康风险。</li>
	<li>通过腔室塞的进样口加入 5 μL 0.1 mM 鱼藤酮并记录呼吸，直到获得稳定的信号。 信号在图2中2-4分钟（g）后稳定下来。 注意：处理鱼藤酮时要小心，因为它对人类有健康风险。</li>
	<li>通过腔室塞子的进样口加入 1 μL 4 mg/mL 寡霉素并记录呼吸，直到获得稳定的信号。 信号在图2中2-4分钟（h）后稳定下来。 注意：处理寡霉素时要小心，因为它是一种对人类健康构成风险的毒药。</li>
	<li>通过腔室塞子的进样口加入 1 μL 5 mM 抗霉素 A 并记录呼吸，直到获得稳定的信号。 信号在图2中的2-4分钟（i）后稳定下来。 注意：处理抗霉素 A 时要小心，因为它是一种对人类健康构成风险的毒药。</li>
	<li>在评估排除不参与氧化磷酸化的酶的耗氧量时，从所有其他测量值中减去抗霉素 A 值。</li>
	<li>加入 200 mM N，N，N'，N'-四甲基对苯二胺二盐酸盐（TMPD;电子供体）和 800 mM 抗坏血酸，以保持 TMPD 处于还原状态。通过腔室塞子的注射口注入底物并记录呼吸，直到获得稳定的信号。 信号在图2中2-4分钟（j）后稳定下来。</li>
	<li>TMPD 会受到自氧化的影响，因此从复合物 IV 的测量值中减去产生的耗氧量。</li>
	<li>通过腔室塞的进样口加入NaN3≥100mM，并记录呼吸，直到获得稳定的信号。信号在2-4分钟后稳定以抑制复合物IV活性，仅保留TMPD自氧化。 注意：处理叠氮化钠时要小心，因为它是一种对人类健康构成风险的毒药。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65730/65730fig02.jpg"> 图 2：O2 通量的示意图。 图中显示了氧气通量的示意图。从a-k添加抑制剂和底物后，曲线分为不同的阶段。a：内源性呼吸;b： 透化细胞;c：解耦复合物I呼吸;d：耦合复合物I呼吸;e： 氧磷 ;f： CI和CII的最大解偶联活性;g：复合物II的解耦呼吸;h：漏气;i：残余呼吸;j： TMPD的CIV（U） 解偶呼吸与自氧化;k：TMPD的自氧化。 <a href="https://www.jove.com/files/ftp_upload/65730/65730fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
8.柠檬酸合酶活性
<ol><li>将柠檬酸合酶活性作为单独的参数进行测量，并使用它来标准化高分辨率氧谱仪的测量值。</li>
	<li>将分离的PBMC重悬于1mL线粒体呼吸培养基（MIR05）中，并稀释至8×106 个细胞/ mL。</li>
	<li>在液氮中冷冻并储存在-80°C，直到进行实验或测量新鲜细胞。</li>
	<li>准备所需的所有溶液：0.1 M 三乙醇胺 HCl 缓冲液 pH 8.0、1.0 M Tris-HCl 缓冲液 pH=8.1、10% Triton X-100、10 mM 草乙酸盐溶于 0.1 M 三乙醇胺 HCl 缓冲液 pH 8.0、1.01 mM DTNB 溶于 1.0 M Tris-HCl 缓冲液 pH=8.1、乙酰辅酶 A 12.2 mM 双蒸馏 H2O。</li>
	<li>制备含有5,5'-二硫代-双-（2-硝基贝氢酸）（DTNB;0.1mM）、乙酰辅酶A（0.31mM）、EDTA（50μM）、HCl三乙醇胺（5mM）和Tris HCl（0.1M）的反应培养基（表1）。</li>
	<li>用溶解在双蒸馏H2O中的0.5mM草酰乙酸制备起始试剂（表1）。</li>
	<li>在冰上解冻样品，因为如果解冻太快，柠檬酸合酶会不稳定。</li>
	<li>在用多管液管加入 110 μL 反应培养基之前，将 40 μL 样品加入冰上的 96 孔板中。</li>
	<li>将反应介质和样品在培养箱中加热至30°C5分钟。将起始试剂在水浴中加热至30°C5分钟。</li>
	<li>向每个孔中加入 50 μL 带有多管移液器的起始试剂。通过酶标仪在30°C下以412nm的波长测量吸光度20分钟。</li>
</ol>

用于线粒体生物能量学分析的 PBMC 采集和低温储存

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S., Stiles, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+human+samples+for+assessment+of+mitochondrial+bioenergetics:+Gold+standards,+limitations,+and+future+perspectives.">Utilization of human samples for assessment of mitochondrial bioenergetics: Gold standards, limitations, and future perspectives.</a> Life. 11 (9), 949 (2021).</li><li>Schindowski, K., 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=Impact+of+aging.">Impact of aging.</a> NeuroMol Med. 4 (3), 161-177 (2003).</li><li>Migliore, 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=Searching+for+the+role+and+the+most+suitable+biomarkers+of+oxidative+stress+in+Alzheimer's+disease+and+in+other+neurodegenerative+diseases.">Searching for the role and the most suitable biomarkers of oxidative stress in Alzheimer's disease and in other neurodegenerative diseases.</a> Neurobiol Aging. 26 (5), 587-595 (2005).</li><li>Leutz, S., 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=Reduction+of+trophic+support+enhances+apoptosis+in+PC12+cells+expressing+Alzheimer&#8217;s+APP+mutation+and+sensitizes+cells+to+staurosporine-induced+cell+death.">Reduction of trophic support enhances apoptosis in PC12 cells expressing Alzheimer&#8217;s APP mutation and sensitizes cells to staurosporine-induced cell death.</a> J Mol Neurosci. 18 (3), 189-201 (2002).</li><li>Leuner, K., 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=Peripheral+mitochondrial+dysfunction+in+Alzheimer&#8217;s+disease:+Focus+on+lymphocytes.">Peripheral mitochondrial dysfunction in Alzheimer&#8217;s disease: Focus on lymphocytes.</a> Mol Neurobiol. 46 (1), 194-204 (2012).</li><li>Leuner, K., 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=Enhanced+apoptosis,+oxidative+stress+and+mitochondrial+dysfunction+in+lymphocytes+as+potential+biomarkers+for+Alzheimer&#8217;s+disease.">Enhanced apoptosis, oxidative stress and mitochondrial dysfunction in lymphocytes as potential biomarkers for Alzheimer&#8217;s disease.</a> J Neural Transm Suppl. 2007 (72), 207-215 (2007).</li><li>Kartika, R., Wibowo, H., Purnamasari, D., Pradipta, S., Larasati, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Indoleamine+2,3-Dioxygenase+production+and+its+association+to+inflammatory+cytokines+in+peripheral+blood+mononuclear+cells+culture+of+type+2+diabetes+mellitus.">Altered Indoleamine 2,3-Dioxygenase production and its association to inflammatory cytokines in peripheral blood mononuclear cells culture of type 2 diabetes mellitus.</a> Int J Tryptophan Res. 13, 1178646920978236 (2020).</li><li>Cortez-Espinosa, N., 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=CD39+expression+on+Treg+and+Th17+cells+is+associated+with+metabolic+factors+in+patients+with+type+2+diabetes.">CD39 expression on Treg and Th17 cells is associated with metabolic factors in patients with type 2 diabetes.</a> Hum Immunol. 76 (9), 622-630 (2015).</li><li>Mahmoud, F., 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=Effect+of+Diabetea+tea+&#8482;+consumption+on+inflammatory+cytokines+and+metabolic+biomarkers+in+type+2+diabetes+patients.">Effect of Diabetea tea &#8482; consumption on inflammatory cytokines and metabolic biomarkers in type 2 diabetes patients.</a> J Ethnopharmacol. 194, 1069-1077 (2016).</li><li>Volman, J. J., Ramakers, J. D., Plat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+modulation+of+immune+function+by+&#946;-glucans.">Dietary modulation of immune function by &#946;-glucans.</a> Physiol Behav. 94 (2), 276-284 (2008).</li><li>Reddy, M., Eirikis, E., Davis, C., Davis, H. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fuel+feeds+function:+energy+metabolism+and+the+T-cell+response.">Fuel feeds function: energy metabolism and the T-cell response.</a> Nat Rev Immunol. 5 (11), 844-852 (2005).</li><li>Li, P., 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=Mitochondrial+respiratory+dysfunctions+of+blood+mononuclear+cells+link+with+cardiac+disturbance+in+patients+with+early-stage+heart+failure.">Mitochondrial respiratory dysfunctions of blood mononuclear cells link with cardiac disturbance in patients with early-stage heart failure.</a> Sci Rep. 5, 10229 (2015).</li><li>Weiss, S. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+respirometry+to+assess+mitochondrial+function+in+permeabilized+and+intact+cells.">High-resolution respirometry to assess mitochondrial function in permeabilized and intact cells.</a> J Vis Exp. (120), e54985 (2017).</li><li>Wang, W., Zhao, F., Ma, X., Perry, G., Zhu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+dysfunction+in+the+pathogenesis+of+Alzheimer&#8217;s+disease:+recent+advances.">Mitochondria dysfunction in the pathogenesis of Alzheimer&#8217;s disease: recent advances.</a> Mol Neurodegener. 15 (1), 30 (2020).</li><li>Chaturvedi, R. 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作者声明没有利益冲突。

该协议提供了一种以适合生物能量分析的方式从人血液中分离和冷冻保存外周血单核细胞（PBMC）的方法。所描述的方法提供了温和和大量分离PBMC的可能性，具有高活力和足够的细胞用于生物能量测量。它的缺点是，即使中断最小，也会发生长时间的分离，但随后的冷冻保存允许对生物能量学进行与时间无关的测量。使用这种方法，可以在以后的时间点收集和测量样品。它还适用于在多中心试验?...

人外周血单核细胞 （PBMC） 用于许多科学领域的各种应用，包括研究免疫学和生物能量问题，例如与衰老过程或退行性疾病相关的问题 1,2。PBMC 的组成是异质的，由淋巴细胞（B 细胞、T 细胞和 NK 细胞）、单核细胞和树突状细胞组成。这些细胞有时会在受试者中表现出巨大的个体差异和变异，因此需要处理这些细胞的标准化程序。分离物的活力和纯度等重要参数是其处理的基本要求，并且还受到环境因素的影响，例如收集时间、褪黑激素水平、受试者是否禁食等 3,4。
基于对PBMCs生物能量学的研究，我们在此描述了一种适用于其他方法的PBMCs的分离、冷冻保存和培养方法。虽然肌肉活检被认为是线粒体能量代谢的金标准5，但血细胞检查是一种快速、微创的手术。除此之外，越来越多的研究表明，衰老和阿尔茨海默病（AD）中线粒体功能的变化不仅发生在大脑中，还发生在外周6,7,8,9,10。该方法还允许调查其他病症和疾病，包括糖尿病和肥胖症11,12,13。可以分析多发性硬化症患者的基因表达模式，或一般分析免疫功能及其影响14,15,16。
PBMC 通常依靠氧化磷酸化 （OXPHOS） 产生三磷酸腺苷 （ATP）17,18。因此，PBMC 作为替代物涵盖了广泛的应用。在以前的报道中，PBMC 的能量代谢已被用于解决器官功能障碍，例如早期心力衰竭19、感染性休克20 或线粒体功能的性别相关差异4。用于冷冻保存、分离和培养PBMC的通用方法在不同研究所获得的结果的可比性方面具有优势。每个步骤21,22的方案存在很大差异，该方法的目的是为PBMC中的生物能量测量提供指南。
在本文中，我们描述了一种测量PBMC中生物能量参数的方法。我们解释了从人血液中分离、冷冻保存和测量PBMC生物能量学的方法。该方法可用于确定患者的生物能量参数，并在临床环境中对其进行评估。为了应用这些测量方法，研究人员需要访问可以从中获得新鲜血液样本的患者群体。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1 M Triethanolamine-HCl-Buffer (pH = 8,0)</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M Triethanolamine-HCl-Buffer</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 M Tris-HCl-Buffer (pH = 8,1)</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>1.01 mM DTBB</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>10 % Triton X-100</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM Oxalacetat</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>14&#8211;20 G sterile blood draw needles Multi Adapter Sarstedt Safety-Multifly</td>
			<td>Sarstedt</td>
			<td>156353_v</td>
			<td></td>
		</tr>
		<tr>
			<td>37% HCl</td>
			<td>Carl Roth GmbH &#38; Co. KG</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol (EtOH)</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetyl-CoA</td>
			<td>Pancreac Applichem</td>
			<td>A3753</td>
			<td></td>
		</tr>
		<tr>
			<td>ADP</td>
			<td>Sigma-Aldrich</td>
			<td>A5285</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol wipes</td>
			<td></td>
			<td></td>
			<td>&#160;(70% isopropyl alcohol)</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua (bidest.)</td>
			<td>With MilliQ Academic (self-made)</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP-Standard</td>
			<td>Sigma-Aldrich</td>
			<td>6016949</td>
			<td></td>
		</tr>
		<tr>
			<td>Biocoll Seperating Solution</td>
			<td>Biochrom</td>
			<td>6115</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safty cabinet MSC Advantage</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonylcyanid-p-trifluoromethoxy-phenylhydrazon (FCCP)</td>
			<td>Sigma-Aldrich</td>
			<td>C2920</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter TC20 Automated Cell Counter</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Heraeus Megafuge 16 R</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Counting slides, dual chamber for cell counter</td>
			<td>Bio-Rad</td>
			<td>1450016</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotube Cryo.S</td>
			<td>Grainer Bio-One</td>
			<td>126263-2DG</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Sigma-Aldrich</td>
			<td>37008</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxid (DMSO)</td>
			<td>Merck</td>
			<td>102952</td>
			<td></td>
		</tr>
		<tr>
			<td>Disinfection spray</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable gloves latex, rubber, or vinyl.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Distrips (12.5 ml) DistriTips</td>
			<td>Gilson</td>
			<td>F164150</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (DPBS; 10x)</td>
			<td>Gibco (Thermo Scientific)</td>
			<td>15217168</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (EtOH 100%)</td>
			<td>Carl ROTH GmbH &#38; Co. KG</td>
			<td>9065.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr>
			<td>Frezer (-80&#176;C)</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G1626</td>
			<td></td>
		</tr>
		<tr>
			<td>Holder/adapter&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator Midi 40 CO2</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Injection syringe</td>
			<td>Hamilton</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Malate</td>
			<td>Sigma-Aldrich</td>
			<td>M-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>MIR05</td>
			<td>Self-prepared</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty Freezing Container</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td>10110051</td>
			<td></td>
		</tr>
		<tr>
			<td>Multireader CLARIOstar</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen tank Locator 6 plus</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin</td>
			<td>Sigma-Aldrich</td>
			<td>O4876</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxalacetate</td>
			<td>Sigma-Aldrich</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygraph-2k</td>
			<td>Orobororus Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>PAA</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes Performance Pipettor 10 &#956;L, 100 &#956;L, 1000 &#956;L</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell-Park. Memorial-Institute-Medium (RPMI-1640)</td>
			<td>Gibco (Thermo Scientific)</td>
			<td>11530586</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
			<td></td>
		</tr>
		<tr>
			<td>Saccharose</td>
			<td>Carl ROTH GmbH &#38; Co. KG</td>
			<td>9286.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Succinate</td>
			<td>Sigma-Aldrich</td>
			<td>S2378</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylphenylendiamin (TMPD)</td>
			<td>Sigma-Aldrich</td>
			<td>T3134</td>
			<td></td>
		</tr>
		<tr>
			<td>Tourniquet/ Blood pressure cuff</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)amino-methane</td>
			<td>Sigma-Aldrich</td>
			<td>108382</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>108643</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypanblau</td>
			<td>Biochrom</td>
			<td>T6146</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Vaccubrand GmbH &#38; Co.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ViewPlate-96</td>
			<td>Perkin Elmer</td>
			<td>6005181</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath WNB22</td>
			<td>Memmert GmbH &#38; Co. KG</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

cryopreservation and bioenergetic evaluation of human peripheral blood mononuclear cells

真核细胞的生理功能主要依赖于线粒体提供的能量。线粒体功能障碍与代谢疾病和衰老有关。氧化磷酸化起着决定性的作用，因为它对于维持能量稳态至关重要。PBMC 已被确定为测量线粒体功能的微创样本，并已被证明可以反映疾病状况。然而，线粒体生物能量功能的测量可能受到人类样本中几个因素的限制。限制是采集的样本量、采样时间（通常分布在几天内）和位置。对收集的样品进行冷冻保存可以确保样品的一致收集和测量。应注意确保测得的参数在冷冻保存的细胞和新制备的细胞之间具有可比性。在这里，我们描述了从人类血液样本中分离和冷冻保存PBMC的方法，以分析这些细胞中线粒体的生物能量功能。与新鲜收获的细胞相比，根据此处描述的方案冷冻保存的PBMC在细胞数量和活力，三磷酸腺苷水平和测量的呼吸链活性方面仅显示出微小的差异。所述制剂只需要 8-24 mL 人血，因此可以在临床研究期间多中心收集样本并在现场测定其生物能量学。

Human peripheral blood mononuclear cells (PBMCs) are used for various applications in many scientific fields, including the study of immunological and bioenergetic issues, such as those related to aging processes or degenerative diseases1,2. PBMCs are heterogeneous in composition and consist of lymphocytes (B cells, T cells, and NK cells), monocytes, and dendritic cells. The cells sometimes show great individual differences and variations within a subject, so standardized procedures for handling these cells are required. Important parameters such as viability and purity of the isolation are the basic requirements for its handling and are additionally influenced by environmental factors such as the time of collection, the melatonin level, whether the subject is fasting, and others3,4.
Based on studies on bioenergetics of PBMCs, we describe here a method for the isolation, cryopreservation, and cultivation of PBMCs that is suitable for other methods as well. While muscle biopsy is considered the gold standard for mitochondrial energy metabolism5, the examination of blood cells is a rapid, minimally invasive procedure. In addition to this, more and more studies suggest that the changes in mitochondrial function in aging and Alzheimer&#39;s disease (AD) occur not only in the brain but also in the periphery6,7,8,9,10. The method also allows investigations of other conditions and diseases, including diabetes mellitus and obesity11,12,13. Gene expression patterns in multiple sclerosis patients can be analyzed, or immune function and influences on it in general14,15,16.
PBMCs generally rely on oxidative phosphorylation (OXPHOS) to generate adenosine triphosphate (ATP)17,18. Therefore, PBMCs cover a wide range of applications as surrogates. In previous reports, the energy metabolism of PBMCs has been used to address organ dysfunctions, such as in early heart failure19, septic shock20 or sex-associated differences4 in mitochondrial function. A generalized method for cryopreservation isolation and cultivation of PBMCs would have advantages in the comparability of results obtained at different institutes. There is a great deal of variation in the protocols for each step21,22, the goal of this method is to provide a guideline for bioenergetic measurements in PBMCs.
In this article we describe a method for measuring bioenergetic parameters in PBMCs. We explain the methods for isolating, cryopreserving and measuring bioenergetics of PBMCs from human blood. This method can be used to determine bioenergetic parameters in patients and evaluate them in a clinical context. To apply these measurements, researchers need access to a patient population from which fresh blood samples can be obtained.

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 

人外周血单核细胞的冷冻保存与生物能量评价

With the help of this method, we want to describe a procedure which is suitable to cryopreserve PBMCs in such a way that after thawing, the bioenergetic parameters are unchanged. One of the major experimental challenges of cryopreservation is that the freezing and thawing process can damage PBMCs, which affects the following bioenergetic measurements. With our protocol, we try to show a way to preserve PBMCs reliably without damage to the cells so that the measured bioenergetic parameters are not influenced.The possibility of preserving PBMCs allows samples to be collected from different centers and measured at one location. It also makes measurement more time independent. Cryopreservation of PBMCs allows studies to be performed simultaneously at multiple sites where the cells are collected and preserved.The collected samples can then be measured centrally. This streamlines the process and planning of studies.

细胞活力和数量 为了实现成功的分离和冷冻保存，细胞计数和活力应尽可能高。在冷冻保存之前和之后，对细胞进行计数，并确定其活力，以确保细胞的健康和质量。 图 3 是冷冻保存前后 PBMC 的代表性说明，细胞计数和活力几乎没有差异。这表明PBMC已成功分离和保存。
<img alt="Figure 3" class="xfigimg" src="/files...

Watch this Scientific Journal Video about Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation at JoVE.com

分离的外周血单核细胞可用于分析免疫功能和疾病、代谢疾病或线粒体功能。在这项工作中，我们描述了一种从全血制备PBMC和随后冷冻保存的标准化方法。冷冻保存使这个时间和地点独立。

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

在这种方法的帮助下，我们想描述一种适合冷冻保存PBMC的程序，即解冻后生物能量参数保持不变。冷冻保存的主要实验挑战之一是冷冻和解冻过程会损坏PBMC，从而影响以下生物能量测量。通过我们的方案，我们试图展示一种在不损害细胞的情况下可靠地保存PBMC的方法，从而不受测量的生物能量参数的影响。保存PBMC的可能性允许从不同的中心收集样本并在一个位置进行测量。它还使测量与时间更加无关。PBMC的冷冻保存允许在收集和保存细胞的多个部位同时进行研究。然后可以对收集的样品进行集中测量。这简化了研究的过程和计划。

cryopreservation-bioenergetic-evaluation-human-peripheral-blood

Research

JoVE Journal

Immunology and Infection

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

843 次观看.  Justus-Liebig-University of Giessen. 在这种方法的帮助下，我们想描述一种适合冷冻保存PBMC的程序，即解冻后生物能量参数保持不变。冷冻保存的主要实验挑战之一是冷冻和解冻过程会损坏PBMC，从而影响以下生物能量测量。通过我们的方案，我们试图展示一种在不损害细胞的情况下可靠地保存PBMC的方法，从而不受测量的生物能量参数的影响。保存PBMC的可能性允许从不同的中心收集样本并在一个位置进行...

视频: 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 

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.

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.

科研

免疫与感染

Isolation and Cryopreservation of Human Peripheral Blood Mononuclear Cells for Bioenergetic Studies

To begin, add eight milliliters of DPBS into a sterile 50 milliliter conical tube. To this, add eight milliliters of blood and use a three milliliter plastic Pasteur pipette to mix the contents well. Add 15 milliliters of lymphocyte isolation medium into another conical tube.Tilt the tube by 20 to 30 degrees. Then use a three milliliter Pasteur pipette to apply the first layer of the blood DPBS mix gently over the isolation medium. Layer the remaining mixture carefully over the sidewall of the tube.Slowly bring the tube into an upright position to layer the remaining blood onto the blood layer. Centrifuge the tube at 1000G for 10 minutes at room temperature in a swinging bucket with the brakes off. The blood PBMC mix will separate into four distinct layers.With a Pasteur pipette, remove two thirds of the plasma layer, then use a one milliliter pipette to collect the PBMCs. Transfer the PBMCs into a new 50 milliliter tube until the entire layer has been harvested. Then make the volume up to 25 milliliters with DPBS to remove residual isolation medium and other residues.Centrifuge the tube at 100G for 10 minutes at room temperature with the brakes on. Then remove the supernatant with a vacuum pump. Next, resuspend the pellet in one milliliter DPBS and add more DPBS to make up the volume to 25 milliliters to wash and resuspend the pellet.Cool a freezing container to four degrees celsius. Then place fetal bovine serum on ice. Resuspend the cell pellet containing the isolated PBMCs in one milliliter of fetal bovine serum.Next mixed DMSO with cooled serum on ice at a ratio of one to five. Transfer the cell suspension to two milliliter labeled cryotubes, using one tube per collection tube. With an automated cell counter, determine the cell count and viability.Use a one milliliter pipette to drop one milliliter of the DMSOFBS mixture into the tube. Then place the tubes in the pre-cooled freezing container. Place the container in a freezer at minus 80 degrees Celsius for 24 hours.The next day, remove the tubes from the freezer and store them in the gas phase of liquid nitrogen. Cell counts and viability were consistent before and after cryo-preservation indicating successful isolation and preservation.