Raz Zarivach 得到了以色列科学基金会的支持（资助号:163/22）。Tomer Eli Ben Yosef 和 Arie Moran 得到了以色列科学基金会的支持（资助号:2047/20）。我们要感谢丹尼尔·吉特勒（Daniel Gitler）及其在本古里安大学（Ben-Gurion University）的团队的合作、支持和专业知识。

1. 细胞转染
<ol><li>在补充有10%胎牛血清（FBS），2mM L-谷氨酰胺和1x青霉素/链霉素（参见 材料表）的Dulbecco改良Eagle培养基（DMEM）中培养HEK293T细胞在37°C / 5%CO2 的湿润培养箱中培养，直到汇合在10cm平板上（8.8×106 个细胞）。</li>
	<li>将一个 13 mm 盖玻片放入 12 孔板的每个孔中。在 12 mL 完全 DMEM 中稀释步骤 1.1 中的 0.44 x10 6 个胰蛋白酶消化细胞。通过上下移液三到五次充分混合。用 1 mL 混合溶液填充每个孔。在37°C / 5%CO2 的潮湿培养箱中生长过夜。</li>
	<li>用每个孔中 1 mL 无血清 DMEM（参见 材料表）替换每个孔中的完整 DMEM。如步骤1.1所示，将12孔板放回加湿的培养箱中。</li>
	<li>对于每个转染孔，在 1.5 mL 试管中的 100 μL 无血清 DMEM 中稀释 1 μg 含有用 mCherry 荧光蛋白标记的目标蛋白（补充文件 1）的 pAAV2 质粒。</li>
	<li>每次转染将 3 μg 聚乙烯亚胺（PEI，参见 材料表）加入 100 μL 无血清 DMEM 中，每次转染在不同的 1.5 mL 试管中。质粒:PEI比例应为1:3（μg:μg）。</li>
	<li>将步骤1.4和步骤1.5中的两个管涡旋10秒，并在室温下静置至少5分钟。</li>
	<li>将步骤1.4中的一份（每孔100μL）DNA溶液与步骤1.5中的一份（每孔100μL）PEI溶液混合。涡旋最终溶液10秒，并在室温下静置至少20分钟，最多6小时。</li>
	<li>从培养箱中取出 12 孔板。涡旋步骤1.7的最终溶液，并向步骤1.3的12孔板中的每个孔中加入200μL。将 12 孔板放回加湿培养箱（步骤 1.1）。</li>
	<li>3 小时后，用 1 mL 完全 DMEM 替换每个孔中的培养基。如步骤1.1所示，将12孔板放回加湿的培养箱中。</li>
	<li>2天后，从37°C / 5%CO2 培养箱中取出转染的细胞培养物，并使用10倍放大倍率将其置于倒置荧光显微镜上。</li>
	<li>使用显微镜对焦轮，在使用明场光的同时聚焦在细胞上。</li>
	<li>切换到荧光 mCherry 激发 （587 nm） 和发射 （610 nm） 波长，并关闭明场光。检查细胞的荧光以确认ZnT1 mCherry的表达。</li>
	<li>准备染料上样液。<ol><li>从储存在-20°C冰箱中的避光原液中取出4μL锌特异性荧光染料（参见 材料表）的乙酰氧基甲基（AM）酯形式的等分试样，溶解在DMSO（1μg/ μL）中。这种形式允许染料通过简单的扩散通过质膜进入细胞。</li>
		<li>将 4 μL 10% 普鲁酸（或 2 μL 20% 普鲁酸，参见 材料表）加入步骤 1.13.1 的等分试样中。通过上下移液 3 次充分混合溶液，然后将所有 8 μL 加入 1.5 mL 试管中。</li>
		<li>从步骤1.13.2开始，向试管中加入补充有1mg / mL（~0.1%）牛血清白蛋白的750μL林格氏溶液（内部制备，参见 成分表 ），并剧烈涡旋。再加入 750 μL 相同的溶液，再次涡旋以确保最大混合。</li>
	</ol></li>
	<li>将步骤 1.13.3 中的 750 μL 最终溶液加入新的 6 孔培养板中的两个孔中。用铝箔盖住。 注意:为避免漂白，从此步骤开始，6孔板始终用铝箔覆盖。</li>
	<li>使用细镊子，从转染的细胞培养板中取出最多四个重复的盖玻片，并将它们置于步骤1.14中新的6孔板的第一个填充孔中（每孔最多四个载玻片）。对第二个填充孔重复此过程。 注意:同一填充孔中的所有盖玻片必须来自相同的条件。但是，每个井可以包含不同的条件。</li>
	<li>盖上铝箔，轻轻摇晃15-20分钟。</li>
	<li>除去染料上样液，并用新的林格氏加白蛋白洗涤液代替，如步骤1.13.3所示。用铝箔盖住。再次摇晃 20 分钟。这允许细胞内酯酶裂解AM酯。</li>
</ol>2. 显微镜准备
<ol><li>如前所述安排必要的工具（参见 材料表）:能够检测 GFP 和 mCherry 荧光团的倒置荧光显微镜、允许在至少两种溶液之间切换的灌注系统、抽吸系统和灌注室。</li>
	<li>打开显微镜、显微镜的光源和相机。打开抽吸系统。</li>
	<li>清洗灌注系统。用林格氏溶液洗涤第一个腔室，用含有7μM锌溶液的林格氏溶液洗涤第二个腔室，该溶液补充有7μM吡啶硫酮（锌离子载体，参见 材料表）。为避免气泡或间隙，请在每个腔室中留出一些液体。 注意:由于两个容器都连接到连接到灌注室的同一根管子上，因此请始终确保用林格溶液洗涤共享段。</li>
	<li>关闭水龙头，并用林格溶液和林格锌溶液填充适当的腔室，如步骤2.3所示。</li>
</ol>3. 样品制备
<ol><li>取灌注室，将其放置，凹槽的窄边朝上。 注意:凹槽是灌注室中间的一个孔。它允许灌注液进入细胞。凹槽的一侧较窄，另一侧较宽。</li>
	<li>在凹槽周围涂上密封硅胶（参见 材料表）。使用移液器吸头清洁凹槽中的任何密封硅胶。 注意: 确保凹槽周围有足够的硅胶密封件来密封 22 毫米的盖玻片。</li>
	<li>使用细镊子，从洗涤液中取出盖玻片，并将其放在凹槽顶部，细胞朝下。这样，细胞将在实验过程中暴露于灌注凹槽的溶液中。</li>
	<li>将 22 毫米盖玻片放在 13 毫米盖玻片的顶部，然后使用镊子拧紧。注意不要使盖玻片破裂。</li>
	<li>翻转腔室，然后按压它以释放所有存在的液体。用 100 μL 林格氏洗涤液填充凹槽，然后再次按压以确保无泄漏。 注意: 如果检测到泄漏，请在泄漏部位涂抹密封剂，然后重新测试。</li>
	<li>将灌注室安装到平台上，并将其固定。放置灌注管和抽吸管，使灌注在凹槽中的细胞上。</li>
	<li>将灌注速率更改为约 2 mL/min，然后打开林格氏溶液的灌注。确保灌注系统在没有泄漏或溢出的情况下工作。</li>
</ol>4. 测量准备
<ol><li>双击图标打开成像软件（参见 材料表）。使用相关凭据登录。选择连接的相机，然后按 确定。</li>
	<li>使用显微镜左侧的按钮将显微镜放大倍率设置为10倍。</li>
	<li>选择实时取景并使用操纵杆，移动平台以聚焦于凹槽中的单元格。</li>
	<li>灌注打开时，关闭灯，按下界面中的专用按钮将波长更改为 mCherry。使用显微镜对焦轮调整焦距。</li>
	<li>显微镜聚焦在细胞上后，使用操纵杆移动平台以选择合适的细胞贴片。 注意:合适的区域被认为是具有至少 10 个单元格的区域（用于 ROI）和空白区域（用于背景）。</li>
	<li>选择打开/关闭 ROI 下拉菜单，然后选择绘制圆形 ROI。</li>
	<li>用表达mCherry的细胞绘制细胞簇的ROI（指示ZnT1的表达）。</li>
	<li>在与步骤 4.6 相同的工具栏中，单击" 打开/关闭背景 ROI" 按钮，然后将背景 ROI 的位置和大小调整为完全没有单元格的区域。<ol><li>检查 mCherry 和 EGFP 波长，以确保所选背景 ROI 中没有细胞。 注意:调整波长，使mCherry使用520 nm的激发波长和610 nm的发射波长，EGFP使用470 nm的激发波长和520 nm的发射波长。</li>
	</ol></li>
	<li>选择 波长 （λ） 子选项卡。标记其复选框，并确保存在 GFP 波长并标记其复选框。如果没有，请添加并标记它。</li>
	<li>选择 "持续时间 "子选项卡。将测量间隔定义为每 5 秒一次，并更改间隔数以适应实验持续时间。</li>
	<li>在目镜下方显微镜面板上的"对焦"部分，单击"打开"按钮以启用完美对焦系统 （PFS）。</li>
	<li>使用 PFS 对焦轮调整对焦。在软件界面中，在 ND 采集下，确保勾选 PFS on 。</li>
	<li>单击" 立即运行"。</li>
</ol>5.实验步骤
<ol><li>首先使用 Ringer 溶液灌注进行 90 秒基线期测量。</li>
	<li>基线期结束后，关闭林格氏溶液灌注，然后打开林格氏锌溶液灌注。通过单击主界面面板右下角的红旗来标记解决方案的切换。预计会出现荧光的上升。</li>
	<li>一旦荧光上升开始饱和，就用林格氏溶液变回灌注。关闭林格氏锌溶液灌注，然后打开林格氏溶液灌注。</li>
	<li>等到实验时间到期。如果 ZnT1 工作正常，预计荧光会明显而稳定地下降。</li>
</ol>6. 数据导出
<ol><li>要导出带有基线减法的数据，请按减 去基线 按钮，然后在"ND采集窗口"中查看更改。</li>
	<li>单击"ND 采集窗口"软件界面中的 "导出 "按钮。将打开一个 excel 数据表。保存到所需位置。</li>
</ol>7. 数据分析
<ol><li>对于每个 ROI，从前 90-100 秒创建平均基线荧光。</li>
	<li>将为每个 ROI 计算的荧光表示为该 ROI 的背景百分比。</li>
	<li>创建所有 ROI 的行平均值，并将结果绘制为折线图。这将创建所有 ROI 的平均值作为时间的函数。</li>
	<li>使用线性拟合功能，选择用林格氏溶液洗涤后荧光降低的初始速率。方程的斜率值与传输速率相关。</li>
</ol>

使用 FluoZin-3 测定研究哺乳动物细胞中的锌转运机制

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zinc+indicator+FluoZin-3+is+not+perturbed+significantly+by+physiological+levels+of+calcium+or+magnesium.">The zinc indicator FluoZin-3 is not perturbed significantly by physiological levels of calcium or magnesium.</a> Cell Calcium. 44 (4), 422-426 (2008).</li><li>Gee, K. R., Zhou, Z. -. L., Ton-That, D., Sensi, S. L., Weiss, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+zinc+in+living+cells.:+A+new+generation+of+sensitive+and+selective+fluorescent+probes.">Measuring zinc in living cells.: A new generation of sensitive and selective fluorescent probes.</a> Cell Calcium. 31 (5), 245-251 (2002).</li><li>Sensi, S. L., Ton-That, D., Weiss, J. H., Rothe, A., Gee, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mitochondrial+fluorescent+zinc+sensor.">A new mitochondrial fluorescent zinc sensor.</a> Cell Calcium. 34 (3), 281-284 (2003).</li><li>Hessels, A. 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=eZinCh-2:+A+versatile,+genetically+encoded+FRET+sensor+for+cytosolic+and+intraorganelle+Zn2++imaging.">eZinCh-2: A versatile, genetically encoded FRET sensor for cytosolic and intraorganelle Zn2+ imaging.</a> ACS Chemical Biology. 10 (9), 2126-2134 (2015).</li><li>S&#225;nchez-Mart&#237;n, R. M., Cuttle, M., Mittoo, S., Bradley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsphere-based+real-time+calcium+sensing.">Microsphere-based real-time calcium sensing.</a> Angewandte Chemie International Edition. 45 (33), 5472-5474 (2006).</li><li>Namdarghanbari, 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=Reaction+of+the+zinc+sensor+FluoZin-3+with+Zn7-metallothionein:+Inquiry+into+the+existence+of+a+proposed+weak+binding+site.">Reaction of the zinc sensor FluoZin-3 with Zn7-metallothionein: Inquiry into the existence of a proposed weak binding site.</a> Journal of Inorganic Biochemistry. 104 (3), 224-231 (2010).</li><li>Devinney, M. J., Reynolds, I. J., Dineley, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+of+intracellular+free+calcium+and+zinc+using+fura-2FF+and+FluoZin-3.">Simultaneous detection of intracellular free calcium and zinc using fura-2FF and FluoZin-3.</a> Cell Calcium. 37 (3), 225-232 (2005).</li></ol>

作者声明没有利益冲突。

上述方法允许以高时间分辨率直接测量细胞内锌浓度。与其他方法相比，这种涉及监测细胞内Zn2+变化的方法可以显着降低背景噪声。此外，染料对锌的选择性消除了与其他金属阳离子的潜在交叉相互作用18,19。最后，它没有直接的细胞毒性，可以测试活细胞过程19。
但是，这种方法有一定的局限性。首先?...

锌是细胞环境中必需的微量元素。它包含所有蛋白质的三分之一，并参与各种细胞过程，例如催化1、转录2 和结构基序3。然而，尽管是氧化还原惰性的，但高浓度的锌对细胞有毒，这就是为什么没有哺乳动物生物体在没有调节锌稳态的机制的情况下生存的原因。在哺乳动物中，有三种机制负责这一过程:（1）金属硫蛋白，它是富含胞质半胱氨酸的蛋白质，以高亲和力结合锌，从而防止过量的游离胞质锌4;（2） Zrt/Irrt 样蛋白 （ZIP），它们是锌转运蛋白，负责锌通过质膜或细胞内细胞器流入胞质溶胶 4,5,6,7,8;（3） ZnTs，是无处不在的阳离子扩散促进剂 （CDF） 家族的哺乳动物亚群，是锌转运蛋白，因为它们将锌从胞质溶胶挤出穿过质膜或进入细胞内细胞器4,5,6,7,8,9。由于锌对细胞代谢的重要性，了解细胞锌动力学至关重要。
以前评估锌动力学的方法依赖于通过将mRNA与固定组织或细胞的细胞锌测量值相关联来评估不同锌条件下mRNA的表达水平10,11,12。这些方法包括化学检测和免疫组化染色。然而，这些方法只能产生间接测量，因此只能确定细胞内锌浓度与锌转运蛋白表达之间的离线相关性。因此，这些方法无法推断任何需要高时间分辨率的参数。
锌2+ 输运的更直接测量使用锌13的放射性同位素。该方法依赖于放射性标记Zn2+ 的测量来监测锌的传输及其动力学。然而，由于锌对细胞稳态的重要性，多个细胞过程调节细胞内锌浓度。其中包括细胞外结合和几种协同工作以维持细胞内 Zn2+ 水平的严格控制的运输系统。这些过程的结合会产生相当大的背景噪声，这使得测试单个与锌相关的传递函数变得困难。
本文展示了一种通过使用锌特异性荧光染料 FluoZin-3 测量细胞内游离锌浓度来直接监测锌转运速率的方法。该染料对Zn2+ 具有高度特异性，并且几乎不受其他二价阳离子（如钙）的干扰。此外，以酯的形式，它通过非离子扩散进入细胞，然后由于细胞内二酯酶的活性而被捕获。因此，其荧光主要与游离胞质锌浓度相关。这些实验旨在研究锌转运蛋白1（ZnT1）的结构-功能关系，ZnT1是ZnT家族的成员。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm plate</td>
			<td>greiner bio-one</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well cell culture plate</td>
			<td>greiner bio-one</td>
			<td>665180</td>
			<td></td>
		</tr>
		<tr>
			<td>13 mm coverslips</td>
			<td>Superior Marienfeld</td>
			<td>111530</td>
			<td></td>
		</tr>
		<tr>
			<td>22 mm cover slides</td>
			<td>Superior Marienfeld</td>
			<td>101050</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plate</td>
			<td>greiner bio-one</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>bioWorld</td>
			<td>22070008</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride anhydrous, granular</td>
			<td>Sigma Aldrich</td>
			<td>C1016</td>
			<td>Concentration in Ringer solution: 1 mM</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Glentham Life Science</td>
			<td>GC6947</td>
			<td>Concentration in Ringer solution: 10 mM</td>
		</tr>
		<tr>
			<td>Dubelco&#8217;s Modified Eagle Media (DMEM)&#160;</td>
			<td>Sartorius</td>
			<td>01-055-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse Ti inverted microscope</td>
			<td>Nikon</td>
			<td>TI-DH</td>
			<td>Discontinued. Replaced by Eclipse Ti2</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Cytiva</td>
			<td>SH30088.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine tweezers</td>
			<td>Dumont</td>
			<td>0203-55-PS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluozin-3AM</td>
			<td>Invitrogen</td>
			<td>F24195</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone Penicillin-Streptomycin 100x solution</td>
			<td>Cytiva</td>
			<td>SV30010&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>LED illumination system</td>
			<td>CoolLED</td>
			<td>pE-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Biological Industries</td>
			<td>03-020-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Merck</td>
			<td>1.05833</td>
			<td>Concentration in Ringer solution: 0.8 mM</td>
		</tr>
		<tr>
			<td>N[2-Hydroxyethyl]piperazine-N&#39;-[2-ethanesulfonic acid] (HEPES)</td>
			<td>Formedium</td>
			<td>HEPES10</td>
			<td>Concentration in Ringer solution: 10 mM</td>
		</tr>
		<tr>
			<td>Neo 5.5 sCMOS camera</td>
			<td>ANDOR</td>
			<td>DC-152Q-FI</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements imaging software</td>
			<td>Nikon</td>
			<td>AR</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic acid F-127</td>
			<td>Millipore</td>
			<td>540025</td>
			<td></td>
		</tr>
		<tr>
			<td>Pottasium chloride</td>
			<td>Bio-Lab</td>
			<td>163823</td>
			<td>Concentration in Ringer solution: 5.4 mM</td>
		</tr>
		<tr>
			<td>Pyrithione</td>
			<td>Sigma Aldrich</td>
			<td>H3261</td>
			<td>Concentration in Ringer zinc solution: 7 &#956;M</td>
		</tr>
		<tr>
			<td>Silicone Grease Kit</td>
			<td>Warner Instruments</td>
			<td>W4 64-0378</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Bio-Lab</td>
			<td>190305</td>
			<td>Concentration in Ringer solution: 120 mM</td>
		</tr>
		<tr>
			<td>Zinc sulfate</td>
			<td></td>
			<td></td>
			<td>Concentration in Ringer zinc solution: 7 &#956;M</td>
		</tr>
		<tr>
			<td></td>
			<td>Sigma Aldrich</td>
			<td>31665</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing mammalian zinc transporters using an in vitro zinc transport assay

过渡金属（如Zn2+离子）由于其细胞毒性而必须受到严格调控。以前，通过测定不同浓度Zn2+下转运蛋白的表达水平来间接测量Zn2+转运蛋白的活性。这是通过利用免疫组化、测量组织中的 mRNA 或确定细胞 Zn2+ 水平来完成的。随着细胞内Zn 2+传感器的发展，锌转运蛋白的活性目前主要通过关联使用荧光探针检测的细胞内Zn 2+的变化与Zn2+转运蛋白的表达来确定。然而，即使在今天，也只有少数实验室监测细胞内Zn2+的动态变化，并用它来直接测量锌转运蛋白的活性。部分问题在于，在ZnT家族的10种锌转运蛋白中，除了ZnT10（转运锰）外，只有锌转运蛋白1（ZnT1）位于质膜上。因此，将转运活性与细胞内Zn2+浓度的变化联系起来是很困难的。本文介绍了一种使用基于锌特异性荧光染料 FluoZin-3 的测定法确定锌传输动力学的直接方法。这种染料以酯的形式加载到哺乳动物细胞中，然后由于细胞二酯酶活性而被困在胞质溶胶中。细胞利用锌2+ 离子载体吡硫酮加载锌 2+。ZnT1活性从细胞洗脱后荧光减少的线性部分评估。在 470 nm 激发和 520 nm 发射下测量的荧光与游离细胞内 Zn2+ 成正比。选择用mCherry荧光基团标记的表达ZnT1的细胞可以仅监测表达转运蛋白的细胞。该测定用于研究 ZnT1 蛋白的不同结构域对人 ZnT1 转运机制的贡献，ZnT1 是一种从细胞中挤出过量锌的真核跨膜蛋白。

ZnT1 是一种哺乳动物锌转运蛋白，位于细胞质膜上13。它是阳离子扩散促进剂 （CDF） 蛋白家族的成员，可将锌从胞质溶胶挤出到细胞外毫14。ZnT1 具有双结构域结构:跨膜结构域（跨膜结构域）和 C 端结构域14。与其他已知的 CDF 蛋白不同，ZnT1 具有扩展的非结构化 C 末端结构域 （USCTD）。USCTD的作用目前尚不清楚。我们使用上述方案将ZnT1 WT与没?...

Watch this Scientific Journal Video about Exploring Cellular Zinc Regulation Through ZnT1 Functionality at JoVE.com

作者聚焦:通过 ZnT1 功能探索细胞锌调节 

由于与蛋白质功能的因果关系较弱且时间分辨率低，锌转运已被证明具有挑战性。该协议描述了一种通过使用Zn 2 +敏感荧光染料以高时间分辨率监测活细胞中Zn 2 +挤出的方法，从而提供Zn2 +外排的直接测量。

使用 体外 锌转运测定表征哺乳动物锌转运蛋白

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...

主要研究哺乳动物锌转运蛋白的构能关系，特别强调其调控机制。由于锌离子对细胞功能的重要性，因此很难准确测量细胞锌动力学。我们的协议解决了锌动力学的低信噪比和低时间分辨率的问题。该方法允许测试锌流入细胞膜，具有高时间分辨率，并且显着减少背景噪声和其他二价阳离子的交叉相互作用。我们发现哺乳动物转运蛋白 ZnT1 的一个 C 末端结构域的非结构化延伸不参与锌转运功能。这使研究人员能够专注于 ZnT1 蛋白的其他结构域以进行锌转运调节。

characterizing-mammalian-zinc-transporters-using-an-vitro-zinc

Research

JoVE Journal

Biology

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

1.0K 次观看.  Ben-Gurion University. 主要研究哺乳动物锌转运蛋白的构能关系，特别强调其调控机制。由于锌离子对细胞功能的重要性，因此很难准确测量细胞锌动力学。我们的协议解决了锌动力学的低信噪比和低时间分辨率的问题。该方法允许测试锌流入细胞膜，具有高时间分辨率，并且显着减少背景噪声和其他二价阳离子的交叉相互作用。我们发现哺乳动物转运蛋白 ZnT1 的一个 C 末端结构域的非结构?...

视频: 作者聚焦:通过 ZnT1 功能探索细胞锌调节 

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

一个<em在体外 FluoroBlok肿瘤侵袭实验

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

科研

生物学

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

细胞的细胞大分子运输检测在植物利用基因枪法

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

基因组编辑与的CompoZr自锌指核酸酶（ZFNs）

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage.
The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

The DNA-binding protein from starved cells (Dps) plays a crucial role in combating bacterial stress. This article discusses the purification of E. coli Dps and the protocol for an in vitro assay demonstrating Dps-mediated protection of DNA from degradation by reactive oxygen species.

应用的<em在体外</em&gt;保护DNA检测，以可视化应力调解属性的DPS蛋白质

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

锌指核酸酶增强基因打靶的人胚胎干细胞

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

蛋白质直接送货到哺乳动物细胞利用细胞渗透半胱氨酸<sub&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt;锌指域

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

用自动光学相机进行细胞迁移的体外试验优化划痕试验

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

Assessing Mineral Availability in Fish Feeds using Complementary Methods Demonstrated with the Example of Zinc in Atlantic Salmon

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a systematic approach combining different methodologies to assess the availability of zinc (Zn) in Atlantic salmon (Salmo salar). Considering that several Zn chemical species can be present in an Atlantic salmon feed, it was hypothesised that Zn availability is influenced by the Zn chemical species present in the feed. Thus, in this study, the first protocol is about how to extract the different Zn chemical species from the feed and to analyze them by a size exclusion chromatography-inductively coupled plasma mass spectroscopy (SEC-ICP-MS) method. Subsequently, an in vitro method was developed to evaluate the solubility of dietary Zn in Atlantic salmon feeds. The third protocol describes the method to study the impact of changing Zn chemical species composition on the uptake of Zn in a fish intestinal epithelial model using a rainbow trout gut cell line (RTgutGC). Together, the findings from the in vitro methods were compared with an in vivo study examining the apparent availability of inorganic and organic sources of Zn supplemented to Atlantic salmon feeds. The results showed that several Zn chemical species can be found in feeds and the efficiency of an organic Zn source depends very much on the amino acid ligand used to chelate Zn. The findings of the in vitro methods had less correlation with that outcome of the in vivo study. Nevertheless, in vitro protocols described in this article provided crucial information regarding Zn availability and its assessment in fish feeds.

This article explains in detail a systematic approach to assess micro-mineral availability in Atlantic salmon. The methodology includes tools and models with increasing biological complexity: (1) chemical speciation analysis, (2) in vitro solubility, (3) uptake studies in cell lines, and (4) in vivo fish studies.&#160;

使用大西洋鲑鱼锌示例证明的补充方法评估鱼类饲料中的矿物供应情况

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

通过粘附足迹测定法对细胞滚动中的分子粘附进行成像

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous research indicated that multiple pathways could mediate the spontaneous death of neutrophils. A cocktail was developed by simultaneously targeting these pathways, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which prolonged the neutrophil lifespan to greater than 5 days without significantly compromising the neutrophil function. Concurrently, a reliable and stable protocol for assessing and evaluating neutrophil death was also developed. In this work, we show that CLON-G can prolong the neutrophil lifespan in vitro to more than 5 days, and we exhibit the lengthening of the neutrophil lifespan with FACS and confocal fluorescence microscopy. This report introduces procedures for the preparation of CLON-G and showcases an in vitro spontaneous death assay of neutrophils, which can be used for the study of neutrophils and for subsequently interrogating neutrophil death, thus providing a reliable resource for the neutrophil community.

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

This protocol details the preparation of CLON-G to extend the neutrophil lifespan to greater than 5 days and provides a reliable procedure for evaluating neutrophil death with flow cytometry and confocal fluorescence microscopy.

通过 CLON-G 和 体外 自发死亡测定延长中性粒细胞寿命

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous ...

使用 体外 锌转运测定表征哺乳动物锌转运蛋白

使用体外锌转运测定表征哺乳动物锌转运蛋白