这个工作是由来自美国，以色列边防军的赠款，以BS和EDSBS是分子遗传学的希尔达和塞西尔·刘易斯教授主持的一个义不容辞的责任。

注：涉及受试动物的程序获得批准的机构动物护理和使用委员会（IACUC）在魏茨曼科学研究所。 1，样品制备<ol><li>胰腺腺泡隔离<ol><li>在开始之前，请准备300毫升新鲜的Krebs-Ringer碳酸氢盐HEPES（KRBH）中。搭配140毫米的NaCl，4.7 mM的氯化钾，1.16毫米氯化镁2,1毫米氯化钙2，11毫米的葡萄糖，10毫米肝素钠。添加牛血清白蛋白（BSA）和0.1毫克/毫升大豆胰蛋白酶抑制剂（STI）（悬浮介质）。制备5 - 10毫升再悬浮的介质补充有胶原酶（〜0.75毫克/毫升）（消化培养基）。 </li><li>含氧的KRB介质〜30分钟。充氧后加入BSA，防止起泡。把悬浮和消化介质为37℃。注：补氧介质降低细胞的死亡率，并提高响应能力分泌。氧合的效果是明显的，至少6小时。 </li><lI&gt;牺牲鼠标颈椎脱位或CO 2窒息，根据机构的动物护理指南。将动物处死在它的后面，将其附加到夹层垫，清洁腹部表面用乙醇。 注意：为了避免污染，高压灭菌解剖工具和用于在使用前KRBH中的水。过滤器通过0.2微米的过滤器KRBH媒介。之前的胰腺切除冲洗清扫工具用70％的乙醇，并进行解剖上的清洁架。开展3级生物安全柜病毒感染。添加抗生素将O / N培养用培养基（见第4节）。 </li></ol></li><li>小鼠胰腺切除术<ol><li>切口广泛的腹部皮肤和皮下组织层。鉴定肝，胃，肠，胰腺和脾脏（ 图1）。注：在下面的步骤，请小心操作胰腺组织，避免用力过大，机械FORCE可防止自身消化的组织和细胞死亡。 </li><li>通过使用两个钝钳，拉出小肠。开始在小肠近端，从胃的出口附近。从肠中分离出胰腺和在体腔内保持它。一旦到达大肠，发现一个粉红色/白色结缔组织。不要与胰腺混淆。 </li><li>清除尽可能多的胰腺组织尽可能不收取脂肪或结缔组织。由脾胃分离，并切断腹壁血管下方（ 图1A-C）取出胰腺。 注意：由于胰腺腺泡从单一动物的产率是高的，就可以从收集所有的胰腺组织，以防止收集的其他组织避免。 </li></ol></li><li>胶原酶消化<ol><li>冲洗胰腺几次在悬浮介质中加热到37℃，并删除非胰腺其余部分组织。沉浸在〜5毫升预加热消化培养基的胰腺在20ml的闪烁瓶中。切碎胰腺1-3毫米的碎​​片。将它放置在恒温水浴（最好在〜100转的搅拌）。为了加速消化，吸取消化组织，上下每隔几分钟。 注：胶原酶消化的持续时间是主要依赖胶原酶浓度和对机械力施加在组织上的量。采用0.75毫克/毫升胶原酶约15分钟，并吸移该组织与5ml的聚苯乙烯吸管有2mm孔口每3 - 5分钟，产生10的腺泡 - 30个细胞。消化之后，悬挂出现浑浊含有肉眼可见的组织。由于胶原酶效率不同的商业类型和批次间的差异，决定了消化的时间经验。 </li><li>洗涤和过滤<ol><li>转移组织消化到50毫升管和终止挖estion加入45毫升悬浮介质中。通过粗过滤悬浮液（〜1毫米）金属网成新管。离心流过，在100×g下3分钟。 </li><li>弃去上清液并重新悬浮消化的胰腺在50ml悬浮介质中。通过70或100微米的尼龙网过滤悬浮液。离心机和以前一样。 </li><li>为了除去浮，损坏腺泡，弃去上清，重悬在胰腺组织中3 - 5毫升悬浮介质中。申请的胰腺腺泡1毫升在15ml锥形管中填充用7ml悬浮介质中。允许腺泡沉降2 - 3分钟。收集落户在以1毫升吸管的管底的腺泡，并重复这一步骤2 - 3次。 </li><li>查看光或解剖镜下胰腺腺泡来测试自己的病情。在图2中，观察隔离的纯度和损害后的细胞杜里造成吴隔离。立即使用这些腺泡或文化长达20小时。 </li></ol></li></ol></li></ol> 2，淀粉酶分泌测定注：淀粉酶分泌测定可作为胰腺腺泡分泌能力的全球措施。这是通过收集在该胰腺腺泡孵育培养基中，并确定该介质的淀粉酶的活性，通常是通过使用市售的试剂盒来实现的。 <ol><li>确定要检查实验条件的号码（ 如无刺激与刺激）。使用至少三次重复为每个条件。此外，添加一组重复，以确定细胞总淀粉含量（“总”的样品）和一组为中（“零”的样品）的淀粉酶活性。标记的多井板，其中该测定将进行的，并且两组微管对于每个重复的使用。 注：12或24孔板S可以是用于监测1和0.5ml腺泡悬浮液，分别。 </li><li>在30毫升KRBH悬浮培养基洗两次腺泡和在37℃下孵育它们在悬浮培养基中进行30分钟。到多孔板中添加促分泌的各10μl滴达到适当的终浓度。 </li><li>胰腺腺泡分泌到了一个两相的方式渐变回应。具有最佳价值的分泌浓度为50 - 100分胆囊收缩素（CCK）和卡巴的1μM（ 图3A）5。 </li><li>清洗和悬浮在悬浮介质中的腺泡。确保腺泡分布均匀的悬浮体的悬浮液，并等分试样等量入多孔板并进入“total&#39;标记的微管。孵育板30分钟，在37℃。 （最好在温和搅拌水浴（〜50 RPM））。 注：腺泡的确切数量可以experimen之间​​变化TS而应该在每次实验中重复类似。因此，腺泡悬浮液的精确等分是对于不同的重复和条件之间进行比较关键的。 </li><li>在培育期间，产生了&#39;总&#39;和&#39;零&#39;的样本。离心机的&#39;总&#39;微管几秒钟为5000×克，并从上清取出50微升到“zero&#39;标记的微管，并放置在4℃。裂解的腺泡的“总”的样品中添加的Triton X-100的微管，以达到1％的最终浓度。大力旋涡的微管。 注：小份的“总”复制类似实验的重复。裂解的腺泡，导致总淀粉含量的释放。或者，收集腺泡在每种条件和裂解在实验结束时，以确定每个重复的细胞含量。 </li><li>收集的大部分腺泡暂停íNTO第一组中的预标记的微管。离心微管几秒钟为5000×克。将上清液转移到第二组的预标记的微管。 </li><li>测定上层清液的淀粉酶活性用市售的试剂盒。计算淀粉酶的分泌的百分比。 注：这是非常重要的，在阅读其线性范围内淀粉酶的活性。目前淀粉酶的分泌淀粉酶的从初始总含量释放百分比的百分比。减去从各三份培养基的背景淀粉酶水平和由同样归初始总含量划分它。 </li></ol> 3，现场胰腺分泌成像来自胰腺腺泡分泌可以直接通过实时成像进行监控。使用各种荧光染料和传感器，实时成像允许分泌的表征在亚细胞分辨率。 <ol><li>准备新鲜的含氧悬浮介质，因为我n阶1.1并把它带到37℃。置的显微镜，如果配有温度控制器，将其调整到37℃。 </li><li>在30毫升悬浮介质的清洗腺泡细胞，并将其放置在玻璃或塑料滑梯。注意：如果亲脂性染料是要用于标签标记的亲脂性染料质膜，弄脏腺泡不超过5分钟，以防止内部的膜的标签通过内吞作用。在整个工作中，小心操作腺泡和避免过度的移液。 </li><li>一旦在显微镜图像腺泡的形态清晰，避免腺泡这​​显示基底泡或细胞内空泡的成像。添加促分泌逐滴或通过使用灌注腔，过程中或成像的开始之前立即。 注：高倍率镜头具有高数值孔径（ 如 ×63 / 1.4×100 / 1.4）的建议，观察亚细胞结构。 </li></ol> 4。 O /胰腺腺泡N个文化（备用细胞培养技术） 注：O 2 / N培养通常用于腺病毒感染。感染是在10 6 -10 7 PFU / ml的为9 -检查前16小时。 <ol><li>培养胰腺腺泡长达20小时。从一个单一的胰腺在25毫升培养基（DMEM所得悬浮腺泡补充有胎牛血清（FCS）（5％），钠丙酮酸盐（1％），抗生素（1％），L-谷氨酰胺（0.5％），牛血清白蛋白（10毫克/毫升）和STI（0.2毫克/毫升）中，并分成五个10厘米板。杂志征稿与病毒的腺泡在3级生物安全柜中。 </li><li>孵育胰腺腺泡在37℃，5％CO 2的潮湿空气。允许腺泡到后续检查3,8之前，在含氧的再悬浮介质中恢复30分钟。 </li></ol>

<ol>
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The authors declare they have no competing financial interests.

除了 ​​在体外培养，为外分泌腺分泌的研究替代性设置包括从胰管液的活体显微镜和测量。活体显微镜结果表明在小鼠唾液腺10井进行操作。这个方法可以记录分泌实时的完整动物，但在其分辨率和由所述用于操纵外分泌组织被限制。通过从胰管收集体液评估分泌麻醉大鼠11来实现的。然而，由于胰管插管是极其困难的，这种方法很少使用。 这些设?...

外分泌胰腺构成了大部分的哺乳动物胰腺肿块，并致力于生产消化酶1的分泌。外分泌胰腺腺泡中，该功能单元是上皮细胞形成及股关节腔的集群。当激素或神经元的刺激，囊泡充满酶运输到顶端细胞表面，保险丝用它排出它们的内容到管腔1-3。所述分泌的酶，然后通过聚结组导管进入小肠，在那里它们催化的食物分解成的营养成分被排出。 此视频演示如何完好腺泡从整个胰腺被隔离，以及如何其分泌能力进行评估。采用这种设置，胰腺分泌是通过测量淀粉酶刺激后的被释放的相对量进行定量。或者，分泌可活通过使用不同的成像传感器和染料。 胰腺腺泡的离体的设置是有利的，因为分离的腺泡，其保留了完整的胰腺的许多特征，可以被操纵和监控的相比更容易在整个动物4-6。这种设置最初是在上世纪70年代，是因为延长了几个外分泌组织的研究，如唾液和乳腺3-7。值得注意的是，它允许分泌的与这些极化上皮细胞的复杂的细胞组织的干扰最小的研究。

<table border="0" cellpadding="0" cellspacing="0" width="655">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">ICR mice</td>
			<td style="width:115px;">Harlan</td>
			<td style="width:91px;"></td>
			<td style="width:203px;">Any strain will do</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS</td>
			<td>Sigma Aldrich</td>
			<td>H8264</td>
			<td>Can substitude for KRB</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma Aldrich</td>
			<td>A7906</td>
			<td>Fraction V</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin inhibitor</td>
			<td>Sigma Aldrich</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase XI&#160;</td>
			<td>Sigma Aldrich</td>
			<td>C0130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nylon mesh</td>
			<td>BD Falcon</td>
			<td>352360</td>
			<td>70-100&#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amylase infinity reagent&#160;</td>
			<td>Thermo</td>
			<td>TR25421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CCK</td>
			<td>Sigma Aldrich</td>
			<td>C2175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FM4-64</td>
			<td>Lifetechnologies</td>
			<td>F34653</td>
			<td>Diluted to 16&#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20mL scintillation vial</td>
			<td>Sigma Aldrich</td>
			<td>Z190527</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DeltaVision system&#160;</td>
			<td>Applied Precision</td>
			<td></td>
			<td>Consisting of an inverted microscope IX71 equipped with 60x/1.4 or 100x/1.3 objectives (Olympus)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss 510 or 710 confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>60x/1.4 or 100x/1.4 objectives&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Microplate reader</td>
			<td>BioTek</td>
			<td>ELx808</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing the secretory capacity of pancreatic acinar cells

胰腺腺泡细胞产生和分泌的消化酶。这些细胞被组织成一个集群形成和股关节腔内。这项工作表明了这些细胞的分泌能力可以通过分离的腺泡的培养来评估。该设置是有利的，因为分离的腺泡，其保留了完整的胰腺外分泌的许多特征可被操纵和监控的相比更容易在整个动物。胰腺腺泡适当隔离是一个关键要求，以使体外培养将代表腺泡体内自然。该协议演示了如何从小鼠胰腺分离完好腺泡。随后，两个互补的方法来评估胰腺分泌呈现。淀粉酶的分泌试验作为一个全球性的措施，而胰腺分泌的直接成像可以分泌在亚细胞分辨率的表征。总的来说，所述技术presen泰德在这里让实验广谱学习外分泌。

通过透射光观察时进行了适当分离的胰腺腺泡显示一个刻板的形态。其基底领域应该会出现一轮且无泡的。顶域是由几百个分泌囊泡包围并显示在颜色（ 图2A，B）的更暗。细胞核位于基底的水泡区域。细胞碎片和内分泌胰腺，其可以在腺泡隔离（ 图2C，D）的早期阶段被检测到的胰腺导管系统和部件，应该是从最后腺泡悬浮缺席（ 图2A，B）。隔离不当，可能?...

Watch this Scientific Journal Video about Assessing the Secretory Capacity of Pancreatic Acinar Cells at JoVE.com

Assessing the Secretory Capacity of Pancreatic Acinar Cells

孤立的胰腺腺泡保留其在体内的形态和活动，并提供强大的方式进行监控和操作的分泌。这项工作表明如何腺泡可以从小鼠胰腺中分离出来，并且其分泌能力如何进行评估。

评估胰腺腺泡细胞的分泌能力

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work ...

assessing-the-secretory-capacity-of-pancreatic-acinar-cells

Research

JoVE Journal

Biology

16.5K 次观看.  Weizmann Institute of Science. 孤立的胰腺腺泡保留其在体内的形态和活动，并提供强大的方式进行监控和操作的分泌。这项工作表明如何腺泡可以从小鼠胰腺中分离出来，并且其分泌能力如何进行评估。 

视频: Assessing the Secretory Capacity of Pancreatic Acinar Cells

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

人胰腺癌移植瘤的干细胞的分离

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

科研

生物学

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

制备胰腺腺泡细胞钙成像的目的，细胞损伤的测量，以及腺病毒感染

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models.

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

表型鼠标肺功能<em&gt;在体内</em&gt;与肺弥散功能

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods ...

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

人主动脉周脂肪祖细胞的分化能力

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

小鼠胰腺胰岛细胞亚群中的钙动力学成像

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

The Use of the Patch-Clamp Technique to Study the Thermogenic Capacity of Mitochondria

Mitochondrial thermogenesis (also known as mitochondrial uncoupling) is one of the most promising targets for increasing energy expenditure to combat metabolic syndrome. Thermogenic tissues such as brown and beige fats develop highly specialized mitochondria for heat production. Mitochondria of other tissues, which primarily produce ATP, also convert up to 25% of the total mitochondrial energy production into heat and can, therefore, have a considerable impact on the physiology of the whole body. Mitochondrial thermogenesis is not only essential for maintaining the body temperature, but also prevents diet-induced obesity and reduces the production of reactive oxygen species (ROS) to protect cells from oxidative damage. Since mitochondrial thermogenesis is a key regulator of cellular metabolism, a mechanistic understanding of this fundamental process will help in the development of therapeutic strategies to combat many pathologies associated with mitochondrial dysfunction. Importantly, the precise molecular mechanisms that control acute activation of thermogenesis in mitochondria are poorly defined. This lack of information is largely due to a dearth of methods for the direct measurement of uncoupling proteins. The recent development of patch-clamp methodology applied to mitochondria enabled, for the first time, the direct study of the phenomenon at the origin of mitochondrial thermogenesis, H+ leak through the IMM, and the first biophysical characterization of mitochondrial transporters responsible for it, the uncoupling protein 1 (UCP1), specific of brown and beige fats, and the ADP/ATP transporter (AAC) for all other tissues. This unique approach will provide new insights into the mechanisms that control H+ leak and mitochondrial thermogenesis and how they can be targeted to combat metabolic syndrome. This paper describes the patch-clamp methodology applied to mitochondria to study their thermogenic capacity by directly measuring H+ currents through the IMM.

This method article details the main steps in measuring H+ leak across the inner mitochondrial membrane with the patch-clamp technique, a new approach to study the thermogenic capacity of mitochondria.

使用膜片钳技术研究线粒体的产热能力

Mitochondrial thermogenesis (also known as mitochondrial uncoupling) is one of the most promising targets for increasing energy expenditure to combat ...

量化视网膜色素上皮吞噬作用和脂褐素积累

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed lipofuscin. The toxicity of lipofuscin is well established in Stargardt's disease, the most common inherited retinal degeneration, but is more controversial in age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world. Determining lipofuscin toxicity in humans has been difficult, and animal models of Stargardt's have limited toxicity. Thus, in vitro models that mimic human RPE in vivo are needed to better understand lipofuscin generation, clearance, and toxicity. The majority of cell culture lipofuscin models to date have been in cell lines or have involved feeding RPE a single component of the complex lipofuscin mixture rather than fragments/tips of the entire photoreceptor outer segment, which generates a more complete and physiologic lipofuscin model. Described here is a method to induce the accumulation of lipofuscin-like material (termed undigestible autofluorescence material, or UAM) in highly differentiated primary human pre-natal RPE (hfRPE) and induced pluripotent stem cell (iPSC) derived RPE. UAM accumulated in cultures by repeated feedings of ultraviolet light-treated OS fragments taken up by the RPE via phagocytosis. The key ways that UAM approximates and differs from lipofuscin in vivo are also discussed. Accompanying this model of lipofuscin-like accumulation, imaging methods to distinguish the broad autofluorescence spectrum of UAM granules from concurrent antibody staining are introduced. Finally, to assess the impact of UAM on RPE phagocytosis capacity, a new method for quantifying outer segment fragment/tips uptake and breakdown has been introduced. Termed "Total Consumptive Capacity", this method overcomes potential misinterpretations of RPE phagocytosis capacity inherent in classic outer segment "pulse-chase" assays. The models and techniques introduced here can be used to study lipofuscin generation and clearance pathways and putative toxicity.

作者聚焦：改进的脂褐素模型和高度极化人类视网膜色素上皮培养物中外段吞噬作用能力的定量  

This protocol describes a lipofuscin accumulation model in highly differentiated and polarized human retinal pigment epithelial (RPE) cultures and an improved outer segment (OS) phagocytosis assay to detect the total OS consumption/degradation capacity of the RPE. These methods overcome the limitations of previous lipofuscin models and classical pulse-chase outer segment phagocytosis assays.

改进脂褐素模型和高极化人视网膜色素上皮培养物中外段吞噬能力的定量

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging ...