我们感谢东京大学 RCAST 基因组科学和系统生物学和医学实验室的成员。我们特别感谢 Dr. h. Aburatani、Dr. t 儿、(LSBM、RCAST、东京大学)、Dr. k. Tomizuka、Dr. t. 吉田和 Dr. A。 国 (协和发酵麒麟 Co., Ltd.) 进行有益的讨论和支持。这项工作得到了部分支持补助金为年轻科学家 (A) (26710005, 培训), 补助金为创新领域的科学研究 (26116711 和 16H01567, 培训) 和补助金为挑战 日本教育、文化、体育、科学和技术部、武田科学基金会 (培训)、小林癌症研究基金会 (培训) 和癌症研究项目的探索性研究 (16K14605, 培训)治疗进化 (P 创造) 和创新的癌症控制的实际研究从日本医疗研究和发展机构, 阿曼德 (培训)。

1. 酸性培养基的制备
<ol><li>
制备控制 (ph 值 7.4) 和 low-pH (ph 6.8) 培养基
	<ol>
<li>在500毫升的水中溶解4.75 克无 l-谷氨酰胺的 DMEM 粉。</li>
		<li>在压紧瓶中准备 0.33 M NaHCO3解决方案。 注意: 由于 NaHCO3在加热和热分解时发出 CO2气体, 因此使用压力紧瓶非常重要。</li>
		<li>高压釜介质和溶液。允许这些缓冲器冷却到25° c。</li>
		<li>添加5毫升的200毫米 l-谷氨酰胺, 5 毫升的青霉素-链霉素, 和50毫升的胎儿牛血清到500毫升的 DMEM 没有碳酸氢盐。</li>
		<li>对于控制 (pH 7.4) 介质, 添加2.4 毫升的0.33 米 NaHCO3溶液每100毫升的 DMEM 没有碳酸氢盐。对于 low-pH (pH 6.8) 介质, 在没有碳酸氢盐的 DMEM 中, 每100毫升添加0.6 毫升的0.33 米 NaHCO3溶液。</li>
		<li>在37° c 以下 5% CO2下, 检查 24 h 孵育后的介质 pH 值。 注意: NaHCO3解决方案的数量需要根据介质 pH 值进行调整, 这可能因孵化器和气压的 CO2内容而异。理想情况下, 第一次, 最好用不同的碳酸氢盐浓度来测量中 pH 值, 以确定 NaHCO 的3解决方案中添加到 DMEM 的适当数量。<ol>
<li>从 CO2孵化器中取出培养皿后立即收集培养基, 并使用 ph 计测量 ph 值。如有必要, 在37° c 下使用水浴保持介质温度。独立测量介质 pH 值三次。 注意: NaHCO3解决方案应存储在4° c, 并且每个媒体都应进行新的准备。可用于本研究的商业可用产品列在材料表中。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
乳酸诱导或盐酸诱导的酸性培养基的制备
	<ol>
<li>添加74µL 乳酸溶液或125µL 1 M HCl 到100毫升的控制介质 (在步骤1.1.5 中制备)。</li>
		<li>在37° c 以下 5% CO2下, 检查 24 h 孵育后的介质 pH 值。 注: 乳酸或 HCl 溶液的用量也需要根据培养基 pH 值进行调整, 根据孵化箱和气压中 CO2含量的不同而有所不同。理想的情况是, 最好用连续的乳酸或盐酸浓度来测定培养基 ph 值, 以确定适当的剂量来调节培养基的 ph 值。</li>
	</ol>
</li>
</ol>2. 收获和治疗细胞
<ol><li>开始培养细胞在37° c 以下 5% CO2至少1星期在执行实验之前。 注: 本研究中使用的细胞系列于材料表中。</li>
	<li>通道细胞, 当他们达到 70-90% 70-100。不要让细胞成为汇合。在日常栽培中每 2-3 天改变培养基。</li>
	<li>通过加入5毫升 PBS 清洗细胞。吸取 PBS 和添加分离酶, 如1毫升胰蛋白酶。</li>
	<li>孵育在37° c, 直到细胞被四舍五入, 开始分离。</li>
	<li>将5毫升培养基添加到分离细胞和停用酶。将细胞悬浮液转移至15毫升管, 离心机3分钟, 在 300 x g. 吸取上清和 re-suspend 细胞的培养基。</li>
	<li>使用例和台盼蓝计算活细胞。</li>
	<li>种子 5.0 x 105细胞/10 厘米的菜在10毫升的培养基中。孵育细胞为24小时在37° c 以下 5% CO2。</li>
	<li>吸取培养基, 用5毫升 PBS 洗涤细胞, 在1节中加入10毫升的酸性培养基。孵育细胞为24小时在37° c 以下 5% CO2。</li>
</ol>3. RNA 分离
<ol><li>用5毫升 PBS 培养培养基和洗涤细胞。通过添加 RNA 提取试剂来扰乱细胞 (请参阅材料表)。</li>
	<li>用吸管将 rna 提取试剂取出, 然后将 rna 提取试剂/细胞裂解液放入1.5 毫升管中。室温下保持5分钟。</li>
	<li>添加250µL 氯仿, 并大力摇管约十五年代. 在室温下为5分钟, 离心机在≥8,000 x g 为5分钟。</li>
	<li>小心地用吸管除去水相。留下一些水相。在另一个1.5 毫升管的地方。</li>
	<li>在水相中加入550µL 异丙醇, 轻轻搅拌。在室温下, 以最大转速 (≥20,000 x g) 在4° c 的20分钟内离开离心机5分钟。</li>
	<li>倒入异丙醇, 在 DEPC 处理的水中加入1毫升75% 乙醇。轻轻地混合。离心机在 8000 x g 为5分钟。</li>
	<li>吸管关闭乙醇, 让颗粒风干。</li>
	<li>添加约 15-25 µL (取决于产量) 的 DEPC 处理水的 RNA 颗粒。混合彻底。</li>
	<li>用分光光度计测量 260 nm 的 OD, 测量分离 RNA 的浓度。</li>
</ol>4. cDNA 合成
<ol><li>在 PCR 反应管中结合以下成分: 高达5µg 总 RNA, 1 µL 寡糖 dT (50 µM) 或随机引物组合 (60 µM), 1 µL 10 毫米 dNTP, 和核酸-免费水到总容量14µL。</li>
	<li>使用小型台式离心机 (5 s) 对部件进行混合和简单的离心分离。变性样品 RNA 或底漆为5分钟在65° c。简要 (〜 5 s) 使用一个小的台式离心机, 并把样品迅速在冰上至少1分钟。</li>
	<li>添加以下组件: 4 µL 5x 反向转录缓冲, 1 µL 反转录酶 (200 u/µL), 1 µL 核糖核酸抑制剂 (40 u/µL)。 注: 商业可用套件的示例见材料表
</li>
	<li>盖管, 混合, 然后简要地离心的内容使用一个小的台式离心机 (约 5 s)。孵育混合反应混合物在 50-55 ° c 为10分钟。</li>
	<li>在80° c 下孵育10分钟, 使反应钝化。</li>
</ol>5. 用实时 PCR 技术测定酸反应基因的表达
<ol><li>将以下元件组合在384个反应板中: 1 µL 模板 cDNA, 0.75 µL 10X N ", n-二甲基-n--[4-(E)-(3-甲基-13-噻唑-2-ylidene) 甲基]-1-喹啉-1-ium-2-基]-propylpropane-13-二胺, 0.3 µL 50 µM 向前底漆, 0.3 µL 50 µM 反向底漆, 1 µL Taq 聚合酶, 2.5 µL 10x Taq 聚合酶缓冲, 2.5 µL 20 毫米 dNTP, 16.65 µL 水。 注意: 商业可用工具包的示例显示在材料表中。表 1中显示了底漆的列表。在这一步中还可以使用 96-井反应板。还可以使用其他探头 (例如、Taqman 探头)。</li>
	<li>建立了实时 pcr 系统的实验和以下 pcr 方案:50 ° c 为2分钟, 1 周期;95° c 为 10 min, 1 周期;95° c 为十五年代和60° c 为 1 min, 40 周期;95° c 为十五年代, 1 周期;60° c 为十五年代, 1 周期;95° c 为十五年代, 1 周期。</li>
	<li>把盘子放在 qPCR 的仪器里。启动程序。 注: 上调的 pH 响应基因MSMO1, INSIG1和IDI1可以通过蛋白质水平来衡量。</li>
</ol>

<ol>
	<li>Cairns, R. A., Harris, I. S., Mak, T. W. <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+cancer+cell+metabolism.">Regulation of cancer cell metabolism.</a> Nature reviews. Cancer. 11 (2), 85-95 (2011).</li><li>Chang, C. H., 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=Metabolic+Competition+in+the+Tumor+Microenvironment+Is+a+Driver+of+Cancer+Progression.">Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression.</a> Cell. 162 (6), 1229-1241 (2015).</li><li>Hsu, P. P., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+metabolism:+Warburg+and+beyond.">Cancer cell metabolism: Warburg and beyond.</a> Cell. 134 (5), 703-707 (2008).</li><li>Gerweck, L. E., Seetharaman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+pH+gradient+in+tumor+versus+normal+tissue:+potential+exploitation+for+the+treatment+of+cancer.">Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer.</a> Cancer research. 56 (6), 1194-1198 (1996).</li><li>Webb, B. A., Chimenti, M., Jacobson, M. P., Barber, 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=Dysregulated+pH:+a+perfect+storm+for+cancer+progression.">Dysregulated pH: a perfect storm for cancer progression.</a> Nature Rev. Cancer. 11 (9), 671-677 (2011).</li><li>Gatenby, R. A., Gillies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+cancers+have+high+aerobic+glycolysis?.">Why do cancers have high aerobic glycolysis?.</a> Nature reviews. Cancer. 4 (11), 891-899 (2004).</li><li>McBrian, M. A., 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=Histone+acetylation+regulates+intracellular+pH.">Histone acetylation regulates intracellular pH.</a> Mol Cell. 49 (2), 310-321 (2013).</li><li>Damaghi, M., 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=Chronic+acidosis+in+the+tumour+microenvironment+selects+for+overexpression+of+LAMP2+in+the+plasma+membrane.">Chronic acidosis in the tumour microenvironment selects for overexpression of LAMP2 in the plasma membrane.</a> Nat Commun. 6, 8752 (2015).</li><li>Corbet, C., 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=Acidosis+Drives+the+Reprogramming+of+Fatty+Acid+Metabolism+in+Cancer+Cells+through+Changes+in+Mitochondrial+and+Histone+Acetylation.">Acidosis Drives the Reprogramming of Fatty Acid Metabolism in Cancer Cells through Changes in Mitochondrial and Histone Acetylation.</a> Cell Metab. 24 (2), 311-323 (2016).</li><li>Chen, J. 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=The+genomic+analysis+of+lactic+acidosis+and+acidosis+response+in+human+cancers.">The genomic analysis of lactic acidosis and acidosis response in human cancers.</a> PLoS genetics. 4 (12), e1000293 (2008).</li><li>Kondo, A., 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=Extracellular+Acidic+pH+Activates+the+Sterol+Regulatory+Element-Binding+Protein+2+to+Promote+Tumor+Progression.">Extracellular Acidic pH Activates the Sterol Regulatory Element-Binding Protein 2 to Promote Tumor Progression.</a> Cell Rep. 18 (9), 2228-2242 (2017).</li></ol>

作者野近藤是协和发酵麒麟 Co. 的雇员, Ltd。

在这里, 我们描述了一个简单的酸性 pH 培养系统及其评价过程。结合三种培养基酸化方法,即: 即, 降低碳酸氢盐浓度, 添加乳酸和盐酸, 使我们能够深入研究 pH 反应机制, 并对其他肿瘤的细胞反应进行比较。微条件如缺氧或营养饥饿。
该方法的关键是在培养基中测定碳酸氢盐、乳酸和盐酸的适当浓度。在第一次执行此方法时, 用不同的碳酸氢盐浓度测定中 pH 值对于确定?...

肿瘤微环境在肿瘤进展和癌细胞代谢中起关键作用1,2,3。癌细胞经常暴露在缺氧、养分匮乏和酸性细胞外 pH 等条件下。然而, 在肿瘤进展的作用没有被澄清如在缺氧或营养饥饿的广泛。肿瘤组织中的脂肪细胞可以变为酸性, 达到大约 pH 值 6.84,5。酸性 pH 产生于有氧和厌氧酵排泄质子 (H+) 和乳酸的增殖癌细胞5,6。
最近的研究发现, 酸性的组蛋白脱乙酰, 脂肪酸氧化, 和胞酸性溶酶体适应严重酸性环境7,8,9,10。然而, 细胞外酸化影响肿瘤行为的机制和酸性 pH 肿瘤微环境中关键调控因子的身份尚未完全确定。此外, 一些报告描述了各种酸性 pH 介质使用不清楚的碳酸氢盐, 三, 管道, 和 HEPES 缓冲剂或乳酸和 HCl, 但很少有报告显示稳定的调整介质或全面几种独特酸性培养基的比较7,8,9,10
为了阐明细胞外酸化过程中肿瘤细胞的主要调控因子和代谢变化, 我们建立了一个简单的体外培养模型, 以维持酸性细胞的活性, 并研究了在癌细胞中的作用.使用这种方法, 我们维持了一个酸性培养基与6.8 在37° c 以下 5% CO2, 在培养基中使用还原碳酸氢盐浓度。pH 值7.4 被用作正常和控制介质。在 PANC-1 和 AsPC-1 胰腺癌细胞培养过程中, 培养基 pH 值持续至少 24 h, 并逐渐降低 72 h。由于增强的糖酵解加速了不仅质子的排泄, 而且乳酸7,8,9, 我们还建立了一种模拟乳酸诱导酸中毒的培养方法, 添加乳酸, 而不是减少碳酸氢盐浓度。此外, 在培养基中 HCl 诱导的酸性物质允许我们排除细胞对酸性 pH 培养基反应的可能性, 而不是由于碳酸氢盐浓度的降低。此外, 使用不同浓度的 pH 值为6.4 到7.4 的不同介质, 我们可以对细胞反应的影响程度进行评估。

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium&#8220;Nissui&#8221; 2</td>
			<td>Nissui</td>
			<td>05919</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher&#160;</td>
			<td>10438</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Wako</td>
			<td>191-01305</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine solution</td>
			<td>Thermo Fisher&#160;</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Mixed Solution(Stabilized)</td>
			<td>Nacalai</td>
			<td>09367-34</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5g/l-Tripsin/0.53mmol/l-EDTA Solution, with Phenol Red</td>
			<td>Nacalai</td>
			<td>32778-05</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5%-Trypan Blue Stain Solution</td>
			<td>Nacalai</td>
			<td>29853-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactic acid solution</td>
			<td>Sigma-Aldrich</td>
			<td>252476</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid solution</td>
			<td>Sigma-Aldrich</td>
			<td>H9892</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Plus Mini Kit</td>
			<td>QIAGEN</td>
			<td>74134</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript IV First-Strand Synthesis System</td>
			<td>Thermo Fisher&#160;</td>
			<td>18091</td>
			<td></td>
		</tr>
		<tr>
			<td>Power SYBR Green PCR Master Mix&#160;</td>
			<td>Applied Biosystems&#160;</td>
			<td>4368702&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PANC-1</td>
			<td>ATCC</td>
			<td>CRL-1469</td>
			<td></td>
		</tr>
		<tr>
			<td>AsPC-1</td>
			<td>ATCC</td>
			<td>CRL-1682</td>
			<td></td>
		</tr>
		<tr>
			<td>KMS-6</td>
			<td>JCRB Cell Bank</td>
			<td>JCRB0432</td>
			<td></td>
		</tr>
		<tr>
			<td>TIG</td>
			<td>JCRB Cell Bank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MRC9</td>
			<td>ATCC</td>
			<td>CCL-212</td>
			<td></td>
		</tr>
		<tr>
			<td>HUVEC</td>
			<td>ATCC</td>
			<td>CRL-1730</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One Microvolume UV-Vis Spectrophotometer with Wi-Fi</td>
			<td>Thermo Fisher</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio 5 Real-Time PCR System</td>
			<td>Thermo Fisher</td>
			<td>CRL-1682</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of an extracellular acidic ph culture system

肿瘤微环境的条件, 如缺氧或营养饥饿, 在癌症进展和恶性肿瘤中起关键作用。然而, 酸性细胞外 pH 在肿瘤侵袭性中的作用及其机制并没有得到广泛的研究, 与缺氧或营养饥饿的情况。此外, 一个明确的培养方法来模拟酸性细胞外肿瘤微环境没有得到充分报道。
在这里, 我们提出了一个简单的体外培养方法, 以保持酸性细胞外 pH 使用还原碳酸氢盐和增加乳酸或 HCl 浓度的培养基。培养基 pH 值维持至少24小时, 并逐渐减少 72 h 以下的培养 PANC-1 和 AsPC-1 胰腺癌细胞。三独特的酸性介质条件在这项研究中高度上调 pH 反应基因, 如MSMO1, INSIG1和IDI1相比, 缺氧或营养饥饿.这些基因的上调可作为酸性 pH 值的标志物。这些简单的技术有助于阐明酸性肿瘤微环境下肿瘤恶性肿瘤的基本机制。因此, 我们的胞外酸性 ph 培养系统能够发现细胞酸性 ph 反应不仅在癌细胞, 而且在初级细胞, 如肾小管细胞, 与其他酸性疾病, 包括糖尿病酮症酸中毒,乳酸酸中毒, 肾小管酸中毒和呼吸道酸中毒。

为了确定适当的碳酸氢盐的浓度, 我们准备 DMEM 在 0-8 毫米 NaHCO3 (最终浓度在培养基中), 并成功地准备的文化介质 pH 范围从 6.4-7.4 (图 1)。我们准备 DMEM 与8毫米 NaHCO3 (pH 7.4) 作为控制介质, 和2毫米 NaHCO3 (ph 6.8) 作为一个介质与酸性 ph 值根据前一份报告指出, 胞外 ph 值达到 ph 6.8 为固体肿瘤4,<sup class="xref...

Watch this Scientific Journal Video about Establishment of an Extracellular Acidic pH Culture System at JoVE.com

Establishment of an Extracellular Acidic pH Culture System

酸性肿瘤微环境在肿瘤进展中起重要作用。为了评估酸性细胞外 pH 对癌细胞的影响, 在体外,建立了简单的酸性培养体系。

胞外酸性 pH 培养体系的建立

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

establishment-of-an-extracellular-acidic-ph-culture-system

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Biology

Unit 1

Fluid, Electrolyte, and Acid-Base Balance

SCIENTISTS IN ACTION

14.5K 次观看.  Kyowa Hakko Kirin Co., Ltd. 酸性肿瘤微环境在肿瘤进展中起重要作用。为了评估酸性细胞外 pH 对癌细胞的影响, 在体外,建立了简单的酸性培养体系。

视频: Establishment of an Extracellular Acidic pH Culture System

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.
 To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 &deg;C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

酸性α-膨大诱导测量植物细胞壁扩展（蠕变）

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

科研

生物学

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

一个 EX - OVO鸡胚胎培养系统适用于成像和显微外科的应用

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

鼠类全胚胎培养方法，使用的肩型瓶培养系统

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

一个<em&gt;体外</em&gt;企业文化系统来研究甲状腺发展

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

在细胞间接触缺失的两个蜂窝型的插入共培养系统的开发

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

用荧光基因编码的 ph 指示剂对果蝇氏小管上皮细胞内 ph 值的光学定量

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Establishment of a Clonal Culture of Unicellular Conjugating Algae

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. Thus, it is extremely important to develop techniques to establish clonal cultures. In this article, we demonstrate the establishment of clonal cultures of a conjugating alga. Water samples are collected from the field. Subsequently, cells are isolated using a glass capillary pipette, placed in media, and grown under conditions suitable for generating a clonal culture.

In this article, we demonstrate the establishment of clonal cultures of unicellular conjugating algal species collected from a natural field site.

单细胞共轭藻类无性系培养的建立

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

使用自动成像系统测量 iPSC 的汇合度。

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

冷冻保存的上皮隐窝仔猪肠道3D类器官的培养和细胞单层的建立

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

用于微生物群多组学的增强细菌细胞外囊泡分离

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

作者聚焦：加速细菌细胞外囊泡分离和异质性的研究 

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.