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>

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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>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium&amp;ldquo;Nissui&amp;rdquo; 2</td><td>Nissui</td><td>05919</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher</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</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</td><td>18091</td><td></td></tr><tr><td>Power SYBR Green PCR Master Mix&amp;nbsp;</td><td>Applied Biosystems</td><td>4368702&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Cell lines&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</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...

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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

14.6K 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

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

体外肠道宿主-微生物界面合成细菌联合的可行性评价

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

科研

生物化学

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader.

一次人气道上皮细胞气道表面液体 pH 值的实时、半自动荧光测量

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

In vitro Monitoring of Extracellular pH in Real-Time

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to chronic inflammation. Without efficient PMN clearance at sites of infiltration, PMN can accumulate and contribute to chronic inflammatory conditions, including the intestinal diseases ulcerative colitis (UC) and Crohn's Disease (CD). The pH in the distal colon in individuals with active UC can range between a pH of 5 and 6, whereas healthy individuals maintain colonic pH in the range of 6.8-7.4. Extracellular pH has been shown to influence both intestinal epithelial cells and the infiltrating immune cells. More specifically, extracellular acidosis significantly impacts PMN. At pH below 6.5, there are increases in the production of H2O2, inhibition of apoptosis, and increases in the functional lifespan of PMN. Given the significant presence of PMN and extracellular acidification at sites of inflammation, we developed a novel model that allows for the monitoring of extracellular pH during PMN transepithelial migration in real time. Here, we describe this model and how it can be utilized to measure both the apical and basal pH during PMN trafficking. This model can be utilized to monitor extracellular pH under a wide range of conditions; including, hypoxia, PMN transepithelial migration, and for extended periods of time.

<meta charset="utf8"></head><body>实时体外监测细胞外 pH 值

This article represents a useful in vitro assay to measure changes in extracellular pH during neutrophil (PMN) transepithelial migration (TEM)

体外 实时监测细胞外 pH 值

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to ...

<meta charset="utf8"></head><body>DHNA 对 植物乳植杆菌 EET 进行计时安培分析

Characterizing Mediated Extracellular Electron Transfer in Lactic Acid Bacteria with a Three-Electrode, Two-Chamber Bioelectrochemical System

Many bacteria perform extracellular electron transfer (EET), whereby electrons are transferred from the cell to an extracellular terminal electron acceptor. This electron acceptor can be an electrode and electrons can be delivered indirectly via a redox-active mediator molecule. Here, we present a protocol to study mediated EET in Lactiplantibacillus plantarum, a probiotic lactic acid bacterium widely used in the food industry, using a bioelectrochemical system. We detail how to assemble a three-electrode, two-chambered bioelectrochemical system and provide guidance on characterizing EET in the presence of a soluble mediator using chronoamperometry and cyclic voltammetry techniques. We use representative data from 1,4-dihydroxy-2-naphthoic acid (DHNA)-mediated EET experiments with L. plantarum to demonstrate data analysis and interpretation. The techniques described in this protocol can open new opportunities for electro-fermentation and bioelectrocatalysis. Recent applications of this electrochemical technique with L. plantarum demonstrated an acceleration of metabolic flux towards producing fermentation end-products, which are critical flavor components in food fermentation. As such, this system has the potential to be further developed to alter flavors in food production or produce valuable chemicals.

<meta charset="utf8"></head><body>用三电极、两室生物电化学系统表征乳酸菌介导的细胞外电子转移

Here we present a protocol for characterizing mediated extracellular electron transfer (EET) in lactic acid bacteria using a three-electrode, two-chamber bioelectrochemical system. We illustrate this method with Lactiplantibacillus plantarum and the redox mediator 1,4-dihydroxy-2-naphthoic acid and provide a thorough description of the electrochemical techniques used to evaluate mediated EET.

用三电极、两室生物电化学系统表征乳酸菌介导的细胞外电子转移

Many bacteria perform extracellular electron transfer (EET), whereby electrons are transferred from the cell to an extracellular terminal electron ...

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its complex nature, and its diverse association with mammalian components. The goal of this protocol is to culture the gut microbiota in vitro, which allows for the study of the gut microbiota dynamics, without having to consider the contribution of the mammalian milieu. Using in vitro culturing technology, the physiological conditions of the gastro intestinal tract are simulated, including parameters such as pH, temperature, anaerobiosis, and transit time. The intestinal surface of the colon is simulated by adding mucin-coated carriers, creating a mucosal phase, and adding further dimension. The gut microbiota is introduced by inoculating with the human fecal material. Upon inoculation with this complex mixture of bacteria, specific microbes are enriched in the different longitudinal (ascending, transverse and descending colons) and transversal (luminal and mucosal) environments of the in vitro model. It is crucial to allow the system to reach a steady state, in which the community and the metabolites produced remain stable. The experimental results in this manuscript demonstrate how the inoculated gut microbiota community develops into a stable community over time. Once steady state is achieved, the system can be used to analyze bacterial interactions and community functions or to test the effects of any additives on the gut microbiota, such as food, food components, or pharmaceuticals.

Here, we present a protocol for culturing the gut microbiota of the colon in vitro, using a series of bioreactors that simulate the physiological conditions of the gastro intestinal tract.

应用先进的体外培养技术研究人肠道微生物群

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its ...

生物工程

Preparation of a High-quality Primary Cell Culture from Fish Pituitaries

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled environment. Despite vast differences in the physiology between mammals and fish, primary cell culture protocols from fish are often based on mammalian culture conditions, often with only minor modifications. The environmental differences affect not only body temperature, but also blood serum parameters such as osmolality, pH, and pH buffer capacity. As cell culture media and similar working solutions are meant to mimic characteristics of the extracellular fluid and/or blood serum to which a cell is adapted, it is crucial that these parameters are adjusted specifically to the animal in question.
The current protocol describes optimized primary culture conditions for medaka (Oryzias latipes). The protocol provides detailed steps on how to isolate and maintain healthy dissociated pituitary cells for more than one week and includes the following steps: 1. the adjustment of the osmolality to the values found in medaka blood plasma, 2. the adjustment of the incubation temperature to normal medaka temperature (here in the aquarium facility), and 3. the adjustment of the pH and bicarbonate buffer to values comparable to other fish species living at similar temperatures. The results presented using the described protocol promote physiologically meaningful results for medaka and can be used as a reference guide by scientists making primary cell cultures from other non-mammalian species.

Here we describe a protocol to prepare and maintain primary pituitary cell cultures from medaka (Oryzias latipes). The optimized conditions in this protocol take important parameters such as temperature, osmolality, and pH into consideration by mimicking the physiological conditions of the fish, thereby enabling physiologically more meaningful results.

从鱼类垂体制备优质原代细胞培养

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled ...

神经科学

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

胞外酸性 pH 培养体系的建立

摘要

探索更多视频

此视频中的章节

体外肠道宿主-微生物界面合成细菌联合的可行性评价

一次人气道上皮细胞气道表面液体 pH 值的实时、半自动荧光测量

体外实时监测细胞外 pH 值

体外实时监测细胞外 pH 值

用三电极、两室生物电化学系统表征乳酸菌介导的细胞外电子转移

用三电极、两室生物电化学系统表征乳酸菌介导的细胞外电子转移

用三电极、两室生物电化学系统表征乳酸菌介导的细胞外电子转移

应用先进的体外培养技术研究人肠道微生物群

从鱼类垂体制备优质原代细胞培养

液泡和胞质pH值的测量

胞外酸性 pH 培养体系的建立

摘要

探索更多视频

此视频中的章节

体外肠道宿主-微生物界面合成细菌联合的可行性评价

一次人气道上皮细胞气道表面液体 pH 值的实时、半自动荧光测量

体外 实时监测细胞外 pH 值

体外 实时监测细胞外 pH 值

用三电极、两室生物电化学系统表征乳酸菌介导的细胞外电子转移

用三电极、两室生物电化学系统表征乳酸菌介导的细胞外电子转移

用三电极、两室生物电化学系统表征乳酸菌介导的细胞外电子转移

应用先进的体外培养技术研究人肠道微生物群

从鱼类垂体制备优质原代细胞培养

液泡和胞质pH值的测量

体外实时监测细胞外 pH 值

体外实时监测细胞外 pH 值