该项目由路易斯安那州理工大学和弗曼大学的启动资金和 AMR 公司分别资助。额外资金由2015教职员进步津贴提供给 AMR 和从南部的伴生的学院, 一个路易斯安那州 EPSCoR Pfund 授予从国家科学基金会和路易斯安那州董事会, 和旅行奖从南方大学。我们感谢 Dr. 大卫尤班克斯 (弗曼) 的帮助, 统计分析和 Mr. 在拍摄过程中 "帮我们一把"。

1. 制作试剂
<ol>
	<li>通过混合6.7 克酵母氮基, 20 克葡萄糖或半乳糖, 20 毫克腺嘌呤硫酸盐 (增加10毫升的 200 mg/100 毫升水溶液), 20 毫克嘧啶 (增加10毫升 200 mg/100 毫升水溶液) 制备葡萄糖和半乳糖培养基, 60 毫克亮氨酸 (6 毫升的 1 g/100 毫升水溶液), 和20毫克组氨酸 (增加2毫升的 1 g/100 毫升水溶液) 在去离子水中的最后体积 1 l 消毒过滤 (0.2 µm 过滤器)。介质可存放在4° c 以下几周。</li>
	<li>在50% 乙醇中溶解无水乙醇中的粉状 E2, 稀释到适当的工作浓度 (227.5 nm 和 #38; 9.75 nm), 以制备雌二醇 (E2) 标准。 注: 如果雌二醇被作为1毫克的小瓶购买, 可直接将1毫升乙醇添加到瓶子中以溶解雌二醇粉。这将产生一个3.67 毫米的股票 #1。 在990μ l 乙醇中稀释10μ l 的库存 #1, 使1毫升的36.71 μ m 的库存 #2。 然后, 稀释10μ l 的库存 #2 990 μ l 50% 乙醇, 使1毫升 367.13 nM 的股票 #3。 为了使工作库存, 稀释619.7 μ l 库存 #3 380.3 μ l 50% 乙醇, 使1毫升的 227.5 nM 工作库存用于创建标准曲线与酵母表达 ERα。 稀释26.56 μ l 库存 #3 973.44 μ l 50% 乙醇, 使1毫升的 9.75 nM 工作库存用于创建标准曲线与酵母表达 er。密封标准与膜和存储在-20 或-70 ° c。 注意: E2 是一种疑似致癌物和生殖毒物。它是有害的, 如果吸入, 吞咽, 或通过皮肤吸收。E2 可能对 breast-feeding 儿童和胎儿造成伤害。E2 对水生生物有毒。使用适当的个人防护 (手套、油烟罩、防尘面罩), 避免孕期和哺乳期的暴露。将 E2 作为危险废物处理, 不要排放到环境中。</li>
	<li>通过混合 8.52 g Na2HPO4来准备 LacZ 缓冲区, 5.52 g NaH2PO4•H2O, 95.21 毫克氯化镁2, 745.5 毫克氯化钾, 2 克 n-lauroylsarcosine 钠盐, 或 ONPG (400 毫克) 或 CPRG (77.7 毫克) 在去离子水中的最终1 l 存储 LacZ 缓冲的容量在20毫升等分在-20 ° c。 注:20 毫升分是足够的一个 96-井板。</li>
	<li>将105.9 克碳酸钠在去离子水中混合, 将碳酸钠制备为 1 l。</li>
	<li>在水中准备1米糖 (德勤)。在-20 ° c 下冻结25µL 等分。 注意: 德勤是一种急性皮肤和眼部刺激性。使用适当的个人防护设备 (手套, 油烟罩, 防尘面罩), 以避免皮肤和眼睛接触, 吸入和摄入。 注: 每个分是足够的一个 96-井板。</li>
</ol>
2. 准备样品和提取控制
<ol>
	<li>选择要测试的示例。 注: 本文介绍十五杆的数据, 包括肥皂、发膏、洗发水、防晒霜、润唇膏、粉底、剃须膏、指甲油和乳液。</li>
	<li>将1克样品与10毫升无水乙醇结合在一个15毫升锥形管中。准备一个由11毫升乙醇在15毫升锥形管中的负萃取控制。根据需要准备复制。 注: 在所提出的实验设计中, 共制备了三等分15杆和三种萃取控制解决方案, 总共产生了48样本, 其中每一个都用4节中的协议进行了三项分析。单板可容纳多达23样品在 triplicates。这一步使用无水乙醇, 因为它容易蒸发。使用负萃取控制, 以验证雌激素不是从锥形管中浸入样品。</li>
	<li>质管的内容为30分钟的组合, 手动晃动和涡流或在多 vortexer 的震动管。</li>
	<li>离心机锥形管在最大允许转速下, 10 分钟醒酒将每管中的上清液通过40µm 的细胞过滤器变成50毫升的锥形管。每50毫升管的空内容变成一个玻璃闪烁瓶。</li>
	<li>在通风罩上开一周的瓶子, 让乙醇完全蒸发。或者, 在通风罩的氮气下或用旋转蒸发器干燥样品。为了避免对光敏感成分的降解, 保护干燥样品免受光照 (例如, 将不透明的织物放在通风罩门上)。</li>
	<li>在1.0 毫升的50% 乙醇和涡质重建样品。</li>
</ol>
3. 培养和传代酵母14
<ol>
	<li>保持活性酵母培养 (重组 W303a 菌株的S. 酵母14) 通过孵化酵母在过滤灭菌葡萄糖培养基在30° c。亚文化酵母每周在新鲜的过滤器-灭菌葡萄糖媒介。</li>
	<li>两个晚上 (约42小时) 之前准备 YES 板, 亚文化酵母通过加入0.1 毫升的活性酵母培养到10毫升的过滤灭菌葡萄糖培养基使用无菌技术。 在30° c 孵育两晚。 如果酵母在无菌250毫升锥形烧瓶中生长为浅的培养物, 则不需要摇动。 注: 酵母也可以储存在冷藏琼脂板几个月。对于较长时间的贮存 (年), 将 O/N 培养物与 15-50% 的无菌甘油、涡旋和冷冻在-70 ° c 相结合。要准备无菌甘油, 用注射器将300µL 甘油转移到 cryovial 和高压釜中, 并松开瓶盖。然后用无菌技术添加1200µL 酵母培养物。</li>
</ol>
4. 准备好板材 (化验的1天)
<ol>
	<li>在无菌烧杯和使用无菌技术, 稀释酵母从步骤3.2 到光学密度0.065 ±0.005 在 610 nm (OD610) 在过滤灭菌半乳糖介质。<ol>
		<li>通过移120μ l 的每一个酵母样品一式一式三份, 通过一个透明的聚苯乙烯板 (板材不需要无菌) 来确认光学密度。 使用平板分光光度计验证稀释的酵母培养物有一个净 od610的0.065 ±0.005 减去 OD610 读数的半乳糖空白从酵母 od610读数, 以确定净 od610的酵母。为每96个井板准备一个至少30毫升稀释酵母的最终体积。 注: 分光光度计过滤器测量595和 610 nm 之间的光度可用于此测量。</li></ol></li></ol>大约 1:6 v: 酵母的 v 的比率: 媒介典型地产生 OD610接近到 0.065, 因此开始通过增加4.5 毫升酵母到30毫升媒介和增加更多酵母或媒介适当地达到 netOD610在0.060 和0.070 之间。
	
	
	<li>使用无菌技术, 根据平板布局 (图 1), 将稀释的酵母悬浮液添加到无菌、聚丙烯、96井微的井中。在移, 保持源酵母悬浮的持续旋转的容器或与吸管混合。对于这一步, 这是有益的使用重复 pipettor 与无菌注射器小费。 注: 聚丙烯板被灭菌两个堆积在铝箔上的叠板所灭菌。 顶板作为样品板的盖子, 可以洗涤, re-autoclaved, 并重复使用。</li>
	<li>根据平板布局 (图 1), 将120µL 过滤器灭菌的半乳糖介质添加到3附加井 (H10-12)。这些水井是媒体的控制, 只考虑媒体的吸收。</li>
	<li>根据板材布局 (图 1), 在含有酵母悬浮液 (A1-G3) 的油井中, 通过连续稀释 triplicates E2 工作标准 (227.5 nm ERα、9.75 nm er), 在每个板块中构造一个标准曲线。 首先通过增加5µL E2 标准 A1 通过 A3。混合微的内容由移, 然后连续稀释这个悬浮通过移动205µL 从井 A1-3 入井 B1-3。重复205µL 通过行 G 的混合和转移。 注: 在连续稀释完成后, 从井中丢弃205μ l G1-3。在这一步的最后, 所有标准井应该包含120μ l。串行稀释将产生表 1中列出的最终 E2 浓度。对于连续稀释步骤, 它是有益的使用多通道 pipettor 与无菌提示。</li>
	<li>根据板材布局 (图 1), 在含有酵母悬浮液 (H1-3) 的车辆控制井中添加5µL 的车辆 (50% 乙醇)。 注: 车辆控制井用于量化与酵母细胞和媒体相关的背景吸收。此外, 通过比较试验井的 OD610值与车辆控制井的对比, 可以检测样品的细胞毒性或促生长作用。</li>
	<li>根据板材布局 (图 1), 在含有酵母悬浮液的油井中添加5µL triplicates 和负萃取控制。 注: 记下每个井中添加的样品或负萃取控制。 要跟踪哪些油井有样品, 哪些井没有, 从盒子里的相同位置开始用一盒新的小贴士, 然后用小贴士作为板块中相应井的位置。一个96井板可以容纳多达23样品一式三份。</li>
	<li>将样品、萃取控制和车辆控制井的内容混合移。按照图 1的图例中所述, 从每个µL 中移除205µL, 将这些井的音量调整为120个。</li>
	<li>用无菌、粘合剂、多孔薄膜封板。标签板和孵化17小时在30° c。在孵化过程中不需要摇动板</li>

5. 加工 YES 板 (化验2天)
<ol>
	<li>在 17 h 孵化在30° c 之后, 从孵化器去除板材。如果板材将被立刻处理, 继续步5.1.1。如果板材将被处理在以后的日期, 继续步5.1.2。<ol>
		<li>使用多通道 pipettor 将每排水井的内容混合, 然后将50µL 的酵母悬浮液从每口井转移到一个清晰、聚苯乙烯、96井微的相应井。 注: 聚苯乙烯板材不需要无菌, 但应该有一个盖子。使用新的吸管提示为每排水井, 以避免 cross-contaminating 井, 虽然如果移横跨 triplicates 与一个8尖端多通道吸管, 提示只需要改变三个套。<ol>
			<li>给新盘子贴上标签。继续执行步骤5.2。</li>
		</ol>
		</li>
		<li>在加工前先将盘子包装好, 用塑料包装。在4° c 下冷藏包裹的盘子 6 d. 之后, 从冰箱中取出盘子, 拆开它们, 并将所有水井的内容按移的步骤5.1.1。继续执行步骤5.2。</li>
	</ol>
	</li>
	<li>增加20µL 1 M 的 LacZ, 以20毫升的解冻, 室温下的缓冲, 最后浓度为1毫米的双制式。混合 LacZ 缓冲井。如果使用多通道吸管, 则将其放入试剂库。 注意: 戴上手套, 在可能的情况下使用德勤。</li>
	<li>使用多通道吸管或重复 pipettor, 增加200µL 的 LacZ 缓冲, 其中包含了 ONPG 或 CPRG 的所有油井的聚苯乙烯板, 并立即测量和记录的 OD610的所有油井使用一个平板分光光度计。根据需要重复额外的盘子。 注意: OD610读数用于 LacZ 计算的分母 (步骤 6.1)。因此, 在移和细胞沉淀或裂解 LacZ 缓冲之前立即获得读数。如果采样井的 od610值明显高于或低于 od 的车辆控制井的610值, 则样本提取物分别是促生长或细胞毒性。LacZ 计算将纠正小差异的细胞密度跨井, 但如果差异超过30% 在任一方向, 然后稀释重组样本 (从步骤 2.6) 额外的50% 乙醇和重新测试样品返回到步骤3。212。</li>
	<li>盖上盖子的盘子。孵育含有酵母的 ERα14在30° c 40 分钟 (对于 ONPG) 或 3 h (CPRG)。孵育含有酵母的 er14在30° c 70 分钟 (对于 ONPG) 或 4 h (CPRG)。 注意: 当使用 CPRG 时, 潜伏期是灵活的;孵化 2-4 h 产生适当的结果与两个受体, 与更长的孵化增加化验灵敏度。</li>
	<li>在孵化板后, 使用多通道吸管或重复 pipettor, 以增加100µL 的碳酸钠, 每井。碳酸钠提高 pH 值并停止β-乳糖反应。</li>
	<li>测量并记录使用平板分光光度计的所有油井的 od405 (对于 ONPG) 或 od574 (对于 CPRG)。</li>
</ol>
6。计算 LacZ 值
注: LacZ 值对每个样本的颜色变化程度进行量化, 并提供一种标准化的方法, 通过核算多个变量 (如、酵母光学密度、介质光学密度和孵育时间) 在单独的分析中比较值 (如果这在同一个板材的井之间不同)12。
<ol>
	<li>计算介质 (半乳糖) 控制井 (H10-12) 的三个 od610值, 并计算车辆 (50% 乙醇) 控制 (H1-3) 的三份 od405或 od574值的方法。使用这些手段和孵化时间 (t) 在几个小时内的板块改变颜色 (步骤 5.5) 计算 LacZ 值 (对应于每井的颜色变化程度) 的所有标准和样品井使用下列公式: 
	<img alt="Equation 1" src="/files/ftp_upload/55754/55754eq1.jpg"> 
	<img alt="Equation 2" src="/files/ftp_upload/55754/55754eq2.jpg">
	<ol>
		<li>或者, 使用附录 1中的自动应用程序来计算标准和示例的 LacZ 值。 注意: 与应用程序一起使用的板布局必须与图 1中所示的板布局相同。如果不使用一些取样井, 则将空井的吸光度读数保留为吸收数据集的位置, 并粘贴到附录 1 LacZ 应用程序中。 这样可以在应用程序中保留板的空间布局, 并确保介质控制井位于其所需位置。 此外, 如果有空单元格, 则不会执行应用程序。</li>
	</ol>
	</li>
	<li>计算每个 E2 标准和每个样本的三 LacZ 值的方法。报告表示 LacZ 值为µL-1h-1。日志-转换每个 E2 标准的浓度, 并生成一个格式化为模板. csv 文件 (附录 2) 的电子表格文档。</li>
</ol>
7. 用4参数逻辑回归法插值样本雌二醇当量 (EEQs)
注: EEQs 将样本 LacZ 值 (颜色变化) 与用 E2 创建的标准曲线的 LacZ 值关联起来。EEQs 从而确定需要多少样本来引出相同的颜色变化响应作为已知浓度的 E2。
<ol>
	<li>要计算测试样本的 EEQs, 首先绘制标准曲线。拟合平均 LacZ 值为 E2 标准 (y-axis) 和相应的对数变换的 E2 浓度 (x 轴) 到一个四参数的逻辑回归模型。</li>
	<li>使用模型为测试样本 LacZ 值插 EEQs。使用统计软件对 EEQs 进行逻辑回归计算, 如附录 2中所述。</li>
</ol>
8. 标准化 EEQs (ng/毫升) 到 PCP 样品质量
<ol>
	<li>为了使 EEQs 与在步骤4.5 中添加到每个井中的 PCP 样品的数量相关, 将 EEQ (ng/毫升) 乘以镀入样品井 (325 µL) 的总体积, 然后除以被提取的样品的体积 (5 µL)。 注意: 这些值代表 EEQ 每毫升提取的样本。如果样品是使用1克的 pcp, 这些价值也代表 EEQ 每克的 pcp (ng/克)。通过这种方式表达, EEQ 值 (ng/克) 可以在不同的杆中进行比较. 本研究中测试的个人护理产品的 EEQ (ng/g) 值显示在表 2中, 在整个协议中收集的示例数据包括在附录 3。</li>
</ol>

<ol>
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作者没有什么可透露的。

"是" 是一种低成本的方法, 用于检测环境样品中的雌激素配体, 如水、食物、植物组织或个人护理产品。这里提供的数据比较了2雌激素受体 (ERα和 er), 2 底物 (ONPG 和 CPRG), 和2时间线 (2 d 协议没有冷藏和七天的协议与制冷), 以测量 estrogenicity 在个人护理产品通过是化验。7 d, 冷藏协议使用 CPRG 和酵母表达 ERα和/或 er 最好的量化 EEQs, 同时也与时间限制所强加的本科生实验室课程, 只满足一次/周的几个 h。事实上, 与2维无冷冻检测法相比, 七天的冷冻试验与平板复制的方差减少有关。此外, 标准曲线的线性部分略有扩展, 用于收集使用冷冻法的数据。标准曲线的线性部分定义了分析检测范围, 是标准曲线中唯一可以用来插入样品 EEQs 的部分。
在所有的试验样品中, 用 CPRG 测定的 EEQs 低于 ONPG。具有较高的消光系数和较低的 Km和 Vmax, CPRG 的灵敏度比 ONPG17高十倍。因此, CPRG 可用于较低浓度, 可用于检测较低量的β-乳糖。由于这些原因, CPRG 通常优先于 ONPG18,19。但是, 更高的基板灵敏度并不能解释用 CPRG 检测到的较低的 EEQ 值。ONPG 检测到的较高的 EEQ 值可能是由于与该检测的矩阵干扰, 如其他作者2,18所述。来自个人护理产品提取物的黄色颜料可以膨胀 EEQ 检测到的 ONPG。其他人则通过在实验设计2中包括颜料控制来规避这个问题, 这种方法使实验中的车牌数增加一倍。当基质干扰可能是有问题的, CPRG 可能是一个更好的基质的是化验, 虽然它是更昂贵, 需要更长的潜伏期比 ONPG。此外, β-乳糖对基底的劈裂引起的颜色变化更剧烈, CPRG, 使学生更容易可视化结果。
米勒et al.(2010), 谁设计了这个协议中使用的酵母, 指出, 酵母表达 er 是30x 更敏感的 17-雌二醇比酵母表达 ERα, 一个发现证实由我们的数据14。除了在质粒建设的潜在细微差别, 米勒et al.(2010) 无法解释14的灵敏度差异。两种质粒结构之间的一个区别是 ERα表达是由半乳糖调节的, 而 er 表达则由葡萄糖或半乳糖调节。在 "YES" 检测中使用的酵母在葡萄糖培养基中培养, 只有在试验开始稀释时才给予半乳糖。因此, 表达 er 的酵母可能在开始检测之前积累更高的受体蛋白拷贝数, 从而赋予雌激素配体更高的敏感性。
ERα表达酵母的低灵敏度可以解释 ERα与 er 相比, estrogenicity 在样品中的 non-detection 率较高。为了增加样品 EEQs 检测的可能性, 用户可以使用不同的萃取溶剂和方法, 或者在酵母中加入更高的样品量。如果使用较高的取样量, 则应调整 E2 标准的浓度, 以便在化验中使用相同数量的标准和样品。添加更多样本的一个限制是酵母只能耐受 6-10% 乙醇, 在孵化温度为 30 Vs. 35 ° c20时具有更好的耐受性。为了控制乙醇对酵母的影响, 调查人员应在平板布局中添加三份 "仅酵母" 井, 并确认这些 "仅酵母" 井的 od610可与车辆控制井的 od610相媲美。在添加 LacZ 缓冲区后。另外, 在乙醇中溶解的样品可以添加到干微板中, 乙醇在加入酵母之前蒸发掉。砜 (亚甲基亚砜) 也被用作样品中的溶剂, 但最终的工作浓度与酵母应限于 1%12。
"YES" 检测是一种用于检测环境样品中 estrogenicity 的强力筛选工具。具体来说, 是检测配体, 结合与雌激素反应元素的核雌激素受体与直接基因表达14。但是, "是" 也有重要的限制。YES 不检测通过非核机制工作的 EEs, 如膜雌激素受体或涉及其他元素的机制, 如激活蛋白 1 (AP-1) 转录因子。此外, 由于酵母与哺乳动物细胞的新陈代谢能力不一样, 是不能检测到需要代谢活化的配体是雌激素。
此外, 是化验不能轻易区分雌激素和抗配体在复杂的样品。相反, 它测量net estrogenicity, 这是雌激素和抗配体的总和效应。为了量化 ER 拮抗剂的浓度或评价混合物的抑菌活性, 该方法可以通过孵育酵母, 同时使用标准的促效酶 (17-雌二醇) 和一系列的测试样本浓度12进行修改。这个过程决定了, 如果在样品中的拮抗剂可以减少激动因素诱发的记者活动, 并提供一个有用的屏幕, 以确定在样品中 ER 拮抗剂的存在。
此处提供的协议可以容纳多种示例类型, 但有些示例可能需要对提取和示例准备步骤进行修改。例如, EEQs 在一些个人护理产品的复制中差别很大。更易变的样品倾向于包含油滴或否则不是完全地均匀的, 表明一个更加亲油性的溶剂例如二乙醚将是有用的。另外, 在个人护理产品中的油和蜡可以被排除在提取样品与50% 乙醇而不是100% 乙醇。50% 乙醇提取物也将捕获更多的水溶性雌激素配体 (例如, 一些颜料)。然而, 50% 的乙醇蒸发速度比100% 乙醇慢, 因此可以延长萃取时间。此外, 一些样品 (如肥皂) 是细胞毒性酵母, 导致减少细胞密度测量 (OD610)。狐狸et al.(2008) 建议, 如果细胞密度差异超过 30%, 与车辆控制井12相比, 应稀释和重新测试此类样品。
如果是用于分析研究目的, 样品池稀释曲线可以测试先发制人确定在步骤4.5 中添加到酵母中的适当数量的提取物。另外, 提取物可以同时在多个卷 (例如, 5 µL 和20µL 提取物添加到酵母在步骤 4.5) 或稀释, 跨越数量级 (如如, 0.2 µL, 2 µL, 和20µL)。"适当" 的提取量是那些识别 estrogenicity 通过匹配 LacZ 值沿线性部分的标准曲线。优化可以防止在酵母中添加过多或过少的样品, 如细胞毒性、假阴性或超过标准曲线的 estrogenicity。如上所述, 在使用不同数量的配体时, 应调整样品和 E2 标准的体积, 使酵母接触到一致的体积和浓度的乙醇或其他车辆的整个板块。
尽管有潜在的假阴性, 是的检测已被确定为3层的内分泌干扰物测试工具 Schug 的et al.(2013), 谁开发了一个全面的分层协议内分泌干扰 (TiPED)21。在本科教育中, 该方法对与内分泌紊乱、细胞培养、受体结合、酶活性、基因工程、统计学和实验设计相关的教学概念有价值。使用该检测方法的学生还要实践基本和广泛适用的实验室技能, 如串行稀释标准;提取样品;制定解决方案;构造和插值标准曲线;计算浓度;制定解决方案;演示无菌技术;培养细胞;识别变量和控制;收集、组织和分析数据;图的构造和解释;使用普通的实验室设备, 如 micropipettors 和光。

酵母雌激素屏幕 (是的) 被广泛用于量化的配体, 模仿雌二醇 (E2 或17雌二醇) 在各种基质, 包括水, 植物组织, 消费品, 食品和食物1,2,3, 4。这些配体统称为 "环境雌激素 (EEs)"。是的最初开发了作为低费用,在体外选择到在体内测试象啮齿目动物子宫化验5,6和彩虹鳟鱼喂养化验7。这些测试的目的是确定产品是否含有刺激或阻断雌激素依赖性机制的化学物质。检测 EEs 是至关重要的, 因为他们可以干扰正常内源性雌激素信号, 特别是在胎儿发育过程中。这种干扰通过增加肥胖、不孕、癌症和认知损失的风险而危及健康8。
尽管 EEs 和其他物对公共卫生的重要性, 但在本科课程中, 内分泌失调并不常见。这一缺陷部分是由于缺乏活动, 说明了所涉及的原则, 同时也可供本科生就读。另外, YES 协议的几个变体存在9、10、11、12、13, 而这种差异性使分析优化耗时在相关技术方面没有专门培训的实验室协调员。最后, 是的化验通常完成超过1长的一天或2连续天与一个 O/N 孵化。这一时间与本科生实验室课程的格式不兼容, 这通常会在几小时内每周举行一次。
针对这些需求, 这份手稿报告了一个优化的 96-良好的协议, 其中包括个人护理产品 (杆)3的乙醇提取方法和6天的制冷步骤, 以适应每周的实验室会议。绝对乙醇是一种多用途的有机溶剂, 可以溶解各种极性和非极性的溶质。此外, 它适合本科课程, 因为它是现成的, 相对无毒, 负担得起, 并与水互溶;它也容易蒸发, 没有特殊的设备。然而, 乙醇是不理想的提取强烈疏水性内分泌干扰物或许多油和蜡, 后两者是共同的成分在杆. 提取效率差会增加假阴性结果的风险。考虑到这一限制, 调查人员应选择提取程序 (例如、乙醇提取或固相萃取) 来处理样本特征并满足研究目标 (研究与本科生教学)。
这是依赖于重组的酿酒酵母最初创建的 Dr. 查尔斯米勒在杜兰大学。请参见米勒et al.(2010) 为工程质粒的完整地图14。酵母转化为这些质粒组成表达人核 ERα或 er (也称为 ESR1 和 ESR2, 分别) 当生长在含有半乳糖 (ERα) 或葡萄糖或半乳糖 (er) 的培养基中。如果媒介中存在着雌激素化学物质, 它们就会与受体结合, 产生配体受体复合物, 激活β-乳糖 (lacZ) 表达, 与雌激素浓度成正比。然后裂解酵母细胞释放积累的β-乳糖。裂解缓冲液中含有 ONPG 或 CPRG, 由β-乳糖切割, 分别产生黄色或红色产物。通过微板分光光度法测量光度, 可以量化色度产品。颜色变化程度与酵母暴露的雌激素配体的浓度成正比。
基板 (CPRG 或 ONPG) 的选择取决于所测试样品所产生的背景吸收的可能性。例如, 植物提取物通常会增加一个黄色色调的媒体, 人为膨胀的 estrogenicity 措施, 如果 ONPG (量化的 405 nm) 被用作β-乳糖基板。与植物提取物, CPRG (定量在574毫微米) 可能是一个更加适当的比色基体。CPRG 比 ONPG 更贵, 但在1/10 的浓度使用。本文介绍了使用 ONPG 和 CPRG estrogenicities 的 PCP 提取物的数量。
使用 ERα和 er 对环境样品进行定量 estrogenicity 是一种比仅使用其中一种受体更全面的方法。在动物中, 这些受体表现为雌激素和抗配体的差异组织分布、调控活动和结合亲和力15。例如, 植物雌典型地束缚 er 更加强烈2, 而合成化学制品可能显示偏爱为 ERα或 er 或能同样同时束缚两个感受器官15。因此, 结合到一个雌激素受体并不一定预测绑定到另一个。
虽然 EEs 在许多消费品 (如如、杀虫剂、洗涤剂、粘合剂、润滑剂、塑料、食品和药品) 以及植物中发现, 但所提供的数据都是通过选择杆获得的. 杆是引人注目的, 容易可提供, 预算友好, 与环境相关的本科学生。学生可以被邀请把他们最喜欢的杆从家里带到实验室进行测试。他们还可以搜索由环境工作组16开发的皮肤深层数据库, 以生成具有高和低毒性分数的杆的假设驱动比较。这样, 学生可以同时开发先进的实验室技能;从事自我导向、探究性学习;并探讨环境科学与健康方面的新问题。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Equipment</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Vortex Mixer (for single or multiple tubes)</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >02-215-365</td>
			<td >Any mixer will suffice.</td>
		</tr>
		<tr >
			<td >Bucket centrifuge</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >75-063-839</td>
			<td >Any bucket centrifuge will suffice if it is capable of centrifuging conical tubes at 4000 rpm.</td>
		</tr>
		<tr >
			<td >Bunsen burner</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >17-012-823</td>
			<td >Any Bunsen burner will suffice, or an alcohol burner (Fisher Scientific 04-245-1) can be used instead.</td>
		</tr>
		<tr >
			<td >Incubator</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >50125590H</td>
			<td >Any incubator will suffice if it is capable of maintaining 30 &#176;C.</td>
		</tr>
		<tr >
			<td >Microplate reader</td>
			<td >BioTek (www.biotek.com)</td>
			<td >EPOCH2</td>
			<td >A different brand of plate reader will suffice if it can measure absorbance at wavelengths of 405, 574, and 610 nm.</td>
		</tr>
		<tr >
			<td >Gen5 microplate reader and imager software</td>
			<td >BioTek (www.biotek.com)</td>
			<td >GEN5</td>
			<td >Software required depends on make and model of plate reader; GEN5 software is intended for use with BioTek plate reader.</td>
		</tr>
		<tr >
			<td >Refrigerator</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >05LREEFSA</td>
			<td >Any refrigerator will suffice if it is capable of maintaining 4 &#176;C.</td>
		</tr>
		<tr >
			<td >Pipettor or PipetAid compatible with 1-10 ml serological pipets</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >13-681-15E</td>
			<td >Any pipettors will suffice if they are compatible with 1- and 10-ml serological pipets.</td>
		</tr>
		<tr >
			<td >P10 micropipettor</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >F144562G</td>
			<td >Any micropipettor will suffice if it is capable of dispensing 5 &#181;l.</td>
		</tr>
		<tr >
			<td >P200 micropipettor</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >F144565G</td>
			<td >Any micropipettor will suffice if it is capable of dispensing up to 200 &#181;l.</td>
		</tr>
		<tr >
			<td >P300 multichannel pipettor</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >FBE1200300</td>
			<td >Any multichannel pipettor will suffice if it is capable of dispensing 50 to 205 &#181;l.</td>
		</tr>
		<tr >
			<td >Repeating pipettor</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >F164001G</td>
			<td >Must be able to deliver 100-320 &#181;l; this pipettor is optional because a multichannel pipettor can be used instead.</td>
		</tr>
		<tr >
			<td >Name</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments</td>
		</tr>
		<tr >
			<td >Supplies</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Sterile 15-ml conical tubes with caps</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >05-539-801</td>
			<td ></td>
		</tr>
		<tr >
			<td >Glass scintillation vials</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >03-337-4</td>
			<td ></td>
		</tr>
		<tr >
			<td >Polystyrene 96-well, flat-bottom microplates with lid (non-sterile)</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >12565501</td>
			<td ></td>
		</tr>
		<tr >
			<td >Polypropylene 96-well, flat-bottom microplates without lid&#160;</td>
			<td >Cole-Parmer (www.coleparmer.com)</td>
			<td >EW-01728-81</td>
			<td ></td>
		</tr>
		<tr >
			<td >100 ml glass beakers</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >02-539H</td>
			<td >Any glass beakers will suffice if they are autoclavable.</td>
		</tr>
		<tr >
			<td >250 ml glass Erlenmeyer flasks</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td>FB500250</td>
			<td >Any glass Erlenmeyer flasks will suffice if they are autoclavable.</td>
		</tr>
		<tr >
			<td >Sterile, adhesive, porous film&#160;</td>
			<td >VWR (www.vwr.com)</td>
			<td >60941-086</td>
			<td ></td>
		</tr>
		<tr >
			<td >Metal forceps</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >12-000-157</td>
			<td >Any metal forceps will suffice.</td>
		</tr>
		<tr >
			<td >Reagent reservoir</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >07-200-127</td>
			<td ></td>
		</tr>
		<tr >
			<td >Filtration units (0.2 &#181;m)</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >09-761-52</td>
			<td ></td>
		</tr>
		<tr >
			<td >Squirt bottle (for ethanol)</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >02-897-10</td>
			<td >Any laboratory squirt bottles will suffice.</td>
		</tr>
		<tr >
			<td >Autoclavable storage bottles</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >06-414-1D</td>
			<td >Any autoclavable glass storage bottles will suffice.</td>
		</tr>
		<tr >
			<td >10 ml glass serological pipets (sterile)</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >13-678-27F</td>
			<td >Any glass serological pipets will suffice.</td>
		</tr>
		<tr >
			<td >1 ml glass serological pipets (sterile)</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >13-678-27C</td>
			<td >Any glass serological pipets will suffice.</td>
		</tr>
		<tr >
			<td >P10 micropipettor tips</td>
			<td >USA Scientific (www.usascientific.com)</td>
			<td >1120-3810</td>
			<td >Any tips will suffice if they are compatible with the micropipettor above.</td>
		</tr>
		<tr >
			<td >P200 micropipettor tips</td>
			<td >USA Scientific (www.usascientific.com)</td>
			<td >1120-8810</td>
			<td >Any tips will suffice if they are compatible with the micropipettor above.</td>
		</tr>
		<tr >
			<td >P300 micropipettor tips</td>
			<td >USA Scientific (www.usascientific.com)</td>
			<td >1120-9810</td>
			<td >Any tips will suffice if they are compatible with the micropipettor above.</td>
		</tr>
		<tr >
			<td >Syringe tips for repeating pipettor</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >F164150G</td>
			<td >Only required if a repeating pipettor is used.</td>
		</tr>
		<tr >
			<td >Sterile petri dishes</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >FB0875712</td>
			<td >Any sterile petri dishes will suffice.</td>
		</tr>
		<tr >
			<td >Parafilm</td>
			<td >Fisher Scientific (www.fishersci.com)</td>
			<td >S37440</td>
			<td ></td>
		</tr>
		<tr >
			<td >Name</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments</td>
		</tr>
		<tr >
			<td >Yeast&#160;</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Saccharomyces cerevisiae strains that lack the TRP1 gene product (e.g. W303a)</td>
			<td >American Type Culture Company (ATCC) (www.ATCC.org)</td>
			<td >MYA-151</td>
			<td >Recombinant yeast are also available upon request from the authors.</td>
		</tr>
		<tr >
			<td >Receptor/reporter plasmid for ER&#945;</td>
			<td >Addgene (www.addgene.org)</td>
			<td >pRR-ERalpha-5Z (Plasmid #23061)&#160;</td>
			<td ></td>
		</tr>
		<tr >
			<td >Receptor/reporter plasmid for ER&#946;</td>
			<td >Addgene (www.addgene.org)</td>
			<td >pRR-ERbeta-5Z (Plasmid #23062)&#160;</td>
			<td ></td>
		</tr>
		<tr >
			<td >Name</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments</td>
		</tr>
		<tr >
			<td >Chemicals</td>
			<td ></td>
			<td></td>
			<td ></td>
		</tr>
		<tr >
			<td >Difco agar</td>
			<td >BD (www.bd.com)</td>
			<td >214530</td>
			<td ></td>
		</tr>
		<tr >
			<td >Dithiothreitol</td>
			<td >Sigma (www.sigmaaldrich.com)</td>
			<td >D0632</td>
			<td >CAUTION:&#160; Dithiothreitol is an acute skin and eye irritant.&#160; Use appropriate personal protection equipment (gloves, fume hood, dust mask) to avoid skin and eye contact, inhalation and ingestion.</td>
		</tr>
		<tr >
			<td >Ortho-nitrophenyl-&#946;-D-galactopyranoside</td>
			<td >Sigma (www.sigmaaldrich.com)</td>
			<td >N1127</td>
			<td ></td>
		</tr>
		<tr >
			<td >Chlorophenol red-&#946;-D-galactopyranoside</td>
			<td >Sigma (www.sigmaaldrich.com)</td>
			<td >10884308001</td>
			<td ></td>
		</tr>
		<tr >
			<td >Yeast nitrogen base</td>
			<td >Sigma (www.sigmaaldrich.com)</td>
			<td>Y0626</td>
			<td ></td>
		</tr>
		<tr >
			<td >Glucose</td>
			<td >Sigma (www.sigmaaldrich.com)</td>
			<td >G8270</td>
			<td ></td>
		</tr>
		<tr >
			<td >Galactose</td>
			<td >Sigma (www.sigmaaldrich.com)</td>
			<td >G5388</td>
			<td ></td>
		</tr>
		<tr >
			<td >Adenine sulfate</td>
			<td >Sigma (www.sigmaaldrich.com)</td>
			<td >A9126</td>
			<td ></td>
		</tr>
		<tr >
			<td >Uracil</td>
			<td >Sigma (www.sigmaaldrich.com)</td>
			<td >U0750</td>
			<td ></td>
		</tr>
		<tr >
			<td >Leucine</td>
			<td >Sigma (www.sigmaaldrich.com)</td>
			<td >L8000</td>
			<td ></td>
		</tr>
		<tr >
			<td >Histidine</td>
			<td >Sigma (www.sigmaaldrich.com)</td>
			<td >H8000</td>
			<td ></td>
		</tr>
		<tr >
			<td >17 &#946;-Estradiol&#160;</td>
			<td >Sigma (www.sigmaaldrich.com)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; MP Biomedicals&#160;</td>
			<td >E8875-250 mg&#160;&#160;&#160;&#160; 0219456401 - 1 mg</td>
			<td >CAUTION:&#160; Estradiol is a suspected carcinogen and reproductive toxicant.&#160; It is harmful if inhaled, swallowed, or absorbed through skin.&#160; Estradiol may cause harm to breast-fed children and fetuses.&#160; Estradiol is very toxic to aquatic life.&#160; Use appropriate personal protection (gloves, fume hood, dust mask) and avoid exposure during pregnancy and lactation.&#160; Estradiol should be disposed of as hazardous waste and not released to the environment.</td>
		</tr>
		<tr >
			<td >100% ethanol</td>
			<td >Pharmco-Aaper (www.pharmcoaaper.com)</td>
			<td >111000200</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sodium phosphate monobasic monohydrate</td>
			<td >Sigma (www.sigmaaldrich.com)</td>
			<td >S9638</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sodium phosphate dibasic (anhydrous)</td>
			<td >Sigma (www.sigmaaldrich.com)</td>
			<td >S0876</td>
			<td ></td>
		</tr>
		<tr >
			<td >Magnesium chloride</td>
			<td >Sigma (www.sigmaaldrich.com)</td>
			<td >M8266</td>
			<td ></td>
		</tr>
		<tr >
			<td >Potassium chloride</td>
			<td >Sigma (www.sigmaaldrich.com)</td>
			<td >P9333</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sarkosyl (N-lauroylsarcosine sodium salt)</td>
			<td >Sigma (www.sigmaaldrich.com)</td>
			<td >L5125</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sodium carbonate</td>
			<td >Sigma (www.sigmaaldrich.com)</td>
			<td >S7795</td>
			<td ></td>
		</tr>
		<tr >
			<td >Name</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments</td>
		</tr>
		<tr >
			<td >Software</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Excel spreadsheet software</td>
			<td >Microsoft&#160;</td>
			<td ></td>
			<td >Excel is convenient spreadsheet software for managing data outputs and generating .csv files for R analysis.</td>
		</tr>
		<tr >
			<td >Java software (required for Appendix 1 application)&#160;</td>
			<td >Oracle Corporation</td>
			<td ></td>
			<td >To calculate LacZ values using the application in Appendix 1, first download free Java software (https://www.java.com/en/download/), then open the LacZ application in Appendix 1.&#160; LacZ results created by the application can be copied by pressing &#34;control (ctrl) + c&#34; on PC keyboards, or &#34;command + c&#34; on Mac keyboards.</td>
		</tr>
		<tr >
			<td >JMP statistical software version 13.0.0</td>
			<td>SAS Institute</td>
			<td ></td>
			<td >Appendix 2 includes directions on using JMP to fit LacZ data to a four-parameter logistic regression curve.&#160; The curve is used to interpolate test sample estradiol equivalents (EEQs), relative to the estradiol standard curve.</td>
		</tr>
		<tr >
			<td >R statistical computing and graphics software</td>
			<td >R Foundation</td>
			<td ></td>
			<td >Free R software can be downloaded from https://www.r-project.org/.&#160; Directions and code for using R to fit LacZ data to a four-parameter logistic regression curve are given in Appendix 2.</td>
		</tr>
	</tbody>
</table>

detecting estrogenic ligands in personal care products using a yeast estrogen screen optimized for the undergraduate teaching laboratory

酵母雌激素屏幕 (YES) 用于检测环境样品中的雌激素配体, 并在内分泌紊乱的研究中得到广泛应用。雌激素配体包括自然和人为的 "环境雌激素" (EEs), 发现在许多消费品, 包括个人护理产品 (杆), 塑料, 杀虫剂和食品。EEs 扰乱人类和其他动物的荷尔蒙信号, 有可能降低生育率和增加疾病风险。尽管 EEs 和其他内分泌干扰化学品 (物) 对公共卫生的重要性, 但在本科课程中, 内分泌紊乱通常不包括在内。这一缺陷部分是由于缺乏相关的实验室活动, 说明了所涉及的原则, 同时也可供本科生就读。本文提出了一个优化的是量化配体的个人护理产品, 结合雌激素受体α (ERα) 和/或β (er)。该方法采用了两个比色衬底 (邻-硝基苯基β-d-乳糖 (ONPG) 或氯酚红β-d-乳糖 (CPRG)), 由β-乳糖, 6 天的冷冻孵化步骤, 以促进本科生实验室课程的使用, LacZ 计算的自动化应用, 以及相关的4参数逻辑回归分析的 R 代码。该协议的目的是让本科生开发和进行实验, 他们筛选的产品, 他们选择的雌激素模拟。在这个过程中, 他们学习内分泌紊乱, 细胞培养, 受体结合, 酶活性, 基因工程, 统计学, 和实验设计。同时, 他们也实践基本和广泛适用的实验室技能, 如: 计算浓度;制定解决方案;演示无菌技术;串行稀释标准;构造和插值标准曲线;识别变量和控制;收集、组织和分析数据;图的构造和解释;使用普通的实验室设备, 如 micropipettors 和光。因此, 在探索环境科学和健康方面的新问题时, 实施这种方法可以鼓励学生从事探究性学习。

使用本协议对15杆的三份样品 estrogenicities 进行了评估。如米勒et al.所指出(2010), 用酵母表达 er 的测定比用酵母表达 ERα (图 2)14的数量级更敏感。因此, 雌激素活性更经常被发现与 er 表达酵母 (表 2)。八杆显示雌激素活性与 er 和5杆表现为雌激素活性与 ERα。发膏、防晒霜、润肤露和润唇膏均为雌激素, 同时, 粉底、剃须膏和指甲油都是 er 的雌激素。七其他杆 (4 洗发香波, 2 肥皂和1唇膏) 不是雌激素与任一受体在被测试的集中。
除了受体敏感度的差异, 每个样本的 EEQs 不同, 取决于在检测中使用的比色基 (ONPG 或 CPRG) (表 2)。除了一个样本, EEQs 测定使用 ONPG 比确定使用 CPRG。此外, 提取复制之间的差异是最低的 EEQs 测量 er 和 CPRG 和最高的 EEQs 确定 ERα和 ONPG (表 2)。
除了比较比色基, 本研究的1目的是测试潜伏期的时间与本科实验室课程的时间表相一致。典型的是化验要求 17 h 孵化后紧接着孵化在 LacZ 缓冲40分钟-4 小时, 一个时间表, 是不可能实现在一个教学实验室限制一个单一的每周会议。为了便于在教学中使用这种方法, 通过在 17 h 潜伏期后引入一个 6 d 制冷期 (步骤 5.1.2), 对该方法进行了改进。标准曲线从冷藏板是可比较的那些从冷冻板材 (图 2 和 #38; 3), 除了参数的标准误差估计使用四参数的逻辑回归模型是所有对于冷藏板比冷冻板更小 (表 3)。因此, 6 维制冷板减少了误差, 提高了 EEQ 估计的准确度。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55754/55754fig1.jpg"> 
图1。微板布局和标准稀释曲线准备的是化验.E2 标准 (浅灰色井)、样品和阴性提取控制 (白色井) 和车 (H1-3) 和半乳糖媒介 (H10-12) 控制 (深灰色井) 全部被测试了三份。E2 标准曲线 (浅灰色水井) 是通过电镀320µL 酵母进入井 A1 通过 A3 和120µL 酵母进入井 B1 通过 G3。然后, 5 µL E2 (227.5 毫微米为酵母那表达 ERα; 9.75 毫微米为酵母那表达 er) 被增加了到酵母在井 A1 通过 A3。酵母 + E2 悬浮被连续稀释通过转移205µL 从每井到它的井下面, 屈服最后 E2 集中列出在表 1。请注意, 在连续稀释过程结束时, 205 µL 必须从井 G1 通过 G3 被丢弃, 以达到每井120µL 的最终体积。通过将每种提取物 (溶解于50% 乙醇) 中的5µL 添加到320µL 酵母中, 制备了样品和负萃取控制井。通过在酵母中添加5µL 50% 乙醇到320µL, 制备了汽车控制井 (H1-3)。所有样品和控制井混合了由移, 并且205µL 被去除了并且被摒弃调整井容量到120µL 每个。最后, 通过将120µL 的半乳糖介质镀到 H10-12 的油井中, 制备出了介质控制。 要使用附录 1中的 LacZ 计算器和附录 2中描述的 R-based 应用程序, E2 标准曲线以及车辆和半乳糖媒体控件必须按所示进行电镀。 只要有电镀的顺序, 就可以在任何一个白色的井中电镀样品和负萃取控制。 <a href="http://ecsource.jove.com/files/ftp_upload/55754/55754fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55754/55754fig2.jpg"> 
图2。通过4参数逻辑回归生成的 YES 板的标准曲线.两个基质 (ONPG 和 #38; CPRG) 测试使用酵母表达的两个人类雌激素受体 (ERα或 er) 两者都没有 (A) 和与 (B) 6 d 冷冻期后, 17 h 孵化与17雌二醇和样品提取物。所有 E2 标准、媒体和车辆控制都进行了三份测试。点代表三 LacZ 值的手段, 其中 LacZ 值反映了β-乳糖诱导的颜色变化程度。逻辑回归模型参数在表 3中列出。ONPG =邻-硝基苯基β-d-乳糖;CPRG = 氯酚红β-d-乳糖。<a href="http://ecsource.jove.com/files/ftp_upload/55754/55754fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55754/55754fig3.jpg"> 
图3。开发的例子是板材.两个基质 (ONPG 和 #38; CPRG) 测试使用酵母表达人类雌激素受体 (ERα或 er), 要么没有 (左) 或与 (右) 六天的冷冻期后, 17 h 孵化与17雌二醇或样品提取物。所有 E2 标准, 媒体和车辆控制在 triplicates 测试。根据图 1, 每个板的顶行中的最高 E2 浓度和控制 (右侧的左侧和半乳糖介质上的50% 乙醇 + 酵母) 排在每个板块的底部。ONPG =邻-硝基苯基β-d-乳糖;CPRG = 氯酚红β-d-乳糖。<a href="http://ecsource.jove.com/files/ftp_upload/55754/55754fig3large.jpg" target="_blank">请点击这里查看更大版本的这个数字。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>标准编号</td>
			<td>[E2] ERα酵母 (pM)</td>
			<td>[E2] er 酵母 (pM)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>3500</td>
			<td>150</td>
		</tr>
		<tr>
			<td>2</td>
			<td>2208</td>
			<td>94。6</td>
		</tr>
		<tr>
			<td>3</td>
			<td>1393</td>
			<td>59。7</td>
		</tr>
		<tr>
			<td>4</td>
			<td>878</td>
			<td>37。6</td>
		</tr>
		<tr>
			<td>5</td>
			<td>554</td>
			<td>23。7</td>
		</tr>
		<tr>
			<td>6</td>
			<td>349</td>
			<td>15</td>
		</tr>
		<tr>
			<td>7</td>
			<td>220</td>
			<td>9.45</td>
		</tr>
	</tbody>
</table>
表1。最终浓度的 E2 标准使用的是。 粉状 E2 溶解在无水乙醇, 然后稀释到工作库存浓度 227.5 nm (为酵母, 表达 ERα) 和 9.75 nm (酵母, 表达 er) 在50% 乙醇。然后, 将工作库存添加到含有半乳糖培养基中酵母的微井中, 然后依次稀释到表中所示的浓度。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">样品测试</td>
			<td colspan="2">化验条件</td>
			<td colspan="4">杆雌激素当量 (EEQs)</td>
		</tr>
		<tr>
			<td>pcp#</td>
			<td>示例类型</td>
			<td>受体</td>
			<td>基</td>
			<td>EEQ 1 (ng/克)</td>
			<td>EEQ 2 (ng/克)</td>
			<td>EEQ 3 (ng/克)</td>
			<td>平均 EEQ (ng/克)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>发膏</td>
			<td>ERα</td>
			<td>onpg</td>
			<td>钕</td>
			<td>0.379</td>
			<td>钕</td>
			<td>和 #60; 0.379</td>
		</tr>
		<tr>
			<td>1</td>
			<td>发膏</td>
			<td>ERα</td>
			<td>CPRG</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
		</tr>
		<tr>
			<td>1</td>
			<td>发膏</td>
			<td>er</td>
			<td>onpg</td>
			<td>0.528</td>
			<td>0.338</td>
			<td>0.363</td>
			<td>0.410</td>
		</tr>
		<tr>
			<td>1</td>
			<td>发膏</td>
			<td>er</td>
			<td>CPRG</td>
			<td>钕</td>
			<td>0.215</td>
			<td>钕</td>
			<td>和 #60; 0.215</td>
		</tr>
		<tr>
			<td>4</td>
			<td>晒</td>
			<td>ERα</td>
			<td>onpg</td>
			<td>6.803</td>
			<td>1.390</td>
			<td>钕</td>
			<td>和 #60; 4.097</td>
		</tr>
		<tr>
			<td>4</td>
			<td>晒</td>
			<td>ERα</td>
			<td>CPRG</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
		</tr>
		<tr>
			<td>4</td>
			<td>晒</td>
			<td>er</td>
			<td>onpg</td>
			<td>1.321</td>
			<td>0.838</td>
			<td>0.818</td>
			<td>0.992</td>
		</tr>
		<tr>
			<td>4</td>
			<td>晒</td>
			<td>er</td>
			<td>CPRG</td>
			<td>0.651</td>
			<td>0.591</td>
			<td>0.725</td>
			<td>0.656</td>
		</tr>
		<tr>
			<td>7</td>
			<td>基础</td>
			<td>ERα</td>
			<td>onpg</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
		</tr>
		<tr>
			<td>7</td>
			<td>基础</td>
			<td>ERα</td>
			<td>CPRG</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
		</tr>
		<tr>
			<td>7</td>
			<td>基础</td>
			<td>er</td>
			<td>onpg</td>
			<td>钕</td>
			<td>0.158</td>
			<td>钕</td>
			<td>和 #60; 0.158</td>
		</tr>
		<tr>
			<td>7</td>
			<td>基础</td>
			<td>er</td>
			<td>CPRG</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
		</tr>
		<tr>
			<td>9</td>
			<td>剃须膏</td>
			<td>ERα</td>
			<td>onpg</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
		</tr>
		<tr>
			<td>9</td>
			<td>剃须膏</td>
			<td>ERα</td>
			<td>CPRG</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
		</tr>
		<tr>
			<td>9</td>
			<td>剃须膏</td>
			<td>er</td>
			<td>onpg</td>
			<td>钕</td>
			<td>0.256</td>
			<td>0.295</td>
			<td>和 #60; 0.276</td>
		</tr>
		<tr>
			<td>9</td>
			<td>剃须膏</td>
			<td>er</td>
			<td>CPRG</td>
			<td>0.507</td>
			<td>0.392</td>
			<td>0.560</td>
			<td>0.486</td>
		</tr>
		<tr>
			<td>10</td>
			<td>指甲油</td>
			<td>ERα</td>
			<td>onpg</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
		</tr>
		<tr>
			<td>10</td>
			<td>指甲油</td>
			<td>ERα</td>
			<td>CPRG</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
		</tr>
		<tr>
			<td>10</td>
			<td>指甲油</td>
			<td>er</td>
			<td>onpg</td>
			<td>0.916</td>
			<td>0.503</td>
			<td>0.554</td>
			<td>0.658</td>
		</tr>
		<tr>
			<td>10</td>
			<td>指甲油</td>
			<td>er</td>
			<td>CPRG</td>
			<td>0.532</td>
			<td>0.628</td>
			<td>0.594</td>
			<td>0.585</td>
		</tr>
		<tr>
			<td>11</td>
			<td>乳液</td>
			<td>ERα</td>
			<td>onpg</td>
			<td>钕</td>
			<td>钕</td>
			<td>2.327</td>
			<td>和 #60; 2.327</td>
		</tr>
		<tr>
			<td>11</td>
			<td>乳液</td>
			<td>ERα</td>
			<td>CPRG</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
		</tr>
		<tr>
			<td>11</td>
			<td>乳液</td>
			<td>er</td>
			<td>onpg</td>
			<td>超出范围</td>
			<td>2.599</td>
			<td>1.845</td>
			<td>和 #62; 2.222</td>
		</tr>
		<tr>
			<td>11</td>
			<td>乳液</td>
			<td>er</td>
			<td>CPRG</td>
			<td>--</td>
			<td>1.986</td>
			<td>1.236</td>
			<td>1.611</td>
		</tr>
		<tr>
			<td>13</td>
			<td>晒</td>
			<td>ERα</td>
			<td>onpg</td>
			<td>14.069</td>
			<td>11.494</td>
			<td>10.189</td>
			<td>11.917</td>
		</tr>
		<tr>
			<td>13</td>
			<td>晒</td>
			<td>ERα</td>
			<td>CPRG</td>
			<td>4.773</td>
			<td>5.790</td>
			<td>5.850</td>
			<td>5.471</td>
		</tr>
		<tr>
			<td>13</td>
			<td>晒</td>
			<td>er</td>
			<td>onpg</td>
			<td>超出范围</td>
			<td>2.580</td>
			<td>超出范围</td>
			<td>和 #62; 2.580</td>
		</tr>
		<tr>
			<td>13</td>
			<td>晒</td>
			<td>er</td>
			<td>CPRG</td>
			<td>0.292</td>
			<td>0.240</td>
			<td>钕</td>
			<td>和 #60; 0.266</td>
		</tr>
		<tr>
			<td>15</td>
			<td>润唇膏</td>
			<td>ERα</td>
			<td>onpg</td>
			<td>钕</td>
			<td>钕</td>
			<td>0.431</td>
			<td>和 #60; 0.431</td>
		</tr>
		<tr>
			<td>15</td>
			<td>润唇膏</td>
			<td>ERα</td>
			<td>CPRG</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
			<td>钕</td>
		</tr>
		<tr>
			<td>15</td>
			<td>润唇膏</td>
			<td>er</td>
			<td>onpg</td>
			<td>1.202</td>
			<td>1.060</td>
			<td>0.887</td>
			<td>1.050</td>
		</tr>
		<tr>
			<td>15</td>
			<td>润唇膏</td>
			<td>er</td>
			<td>CPRG</td>
			<td>0.820</td>
			<td>0.871</td>
			<td>0.851</td>
			<td>0.847</td>
		</tr>
	</tbody>
</table>
表2。EEQs 杆的非零 EEQs。三等分15杆和三萃取控制在无水乙醇中均质化, 蒸发, 并在50% 乙醇中重组, 共48样品, 每一个都是用 "YES" 法分析的。八杆有至少1非零 EEQ, 而7杆未被发现是雌激素在被测试的集中。EEQ 1, EEQ 2, 和 EEQ 3 指每一个 PCP 的三等分, 每一个在一个单独的板块测试三次。值为 EEQ 1, EEQ 2 和 EEQ 3 是 triplicates 的手段为每分。在该检测中使用的酵母表达的 1 2 亚型的人类雌激素受体 (ERα或 er)。两个色度基板, ONPG 和 CPRG, 测试使用冷冻协议。EEQs 是使用四参数的逻辑回归 (R2值≥ 0.98) 的 LacZ 值的每个7浓度的 E2。低于检测限度的测定。--丢失复制。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">化验条件</td>
			<td colspan="5">模型参数</td>
		</tr>
		<tr>
			<td>受体</td>
			<td>基</td>
			<td>拘捕在4° c 为 6 d</td>
			<td>模型 R2</td>
			<td>增长率 (LacZ 单位/ng/毫升)</td>
			<td>拐点 (ng/毫升)</td>
			<td>下渐近线 (LacZ 单位)</td>
			<td>上部渐近线 (LacZ 单位)</td>
		</tr>
		<tr>
			<td>ERα</td>
			<td>onpg</td>
			<td>不</td>
			<td>和 #62; 0.99</td>
			<td>8.548 ±1.633</td>
			<td>-0.512 ±0.025</td>
			<td>-1.623 ±4.408</td>
			<td>128.11 ±6.31</td>
		</tr>
		<tr>
			<td>ERα</td>
			<td>onpg</td>
			<td>是的</td>
			<td>和 #62; 0.99</td>
			<td>7.618 ±0.758</td>
			<td>-0.587 ±0.014</td>
			<td>-8.378 ±2.223</td>
			<td>99.53 ±2.528</td>
		</tr>
		<tr>
			<td>ERα</td>
			<td>CPRG</td>
			<td>不</td>
			<td>和 #62; 0.99</td>
			<td>8.256 ±2.182</td>
			<td>-0.610 ±0.033</td>
			<td>0.853 ±3.333</td>
			<td>62.52 ±3.473</td>
		</tr>
		<tr>
			<td>ERα</td>
			<td>CPRG</td>
			<td>是的</td>
			<td>和 #62; 0.99</td>
			<td>5.068 ±0.616</td>
			<td>-0.523 ±0.022</td>
			<td>-3.147 ±1.946</td>
			<td>68.06 ±3.069</td>
		</tr>
		<tr>
			<td>er</td>
			<td>onpg</td>
			<td>不</td>
			<td>和 #62; 0.99</td>
			<td>1.041 ±2.004</td>
			<td>-0.898 ±4.410</td>
			<td>-32.98 ±86.62</td>
			<td>248.6 ±778。9</td>
		</tr>
		<tr>
			<td>er</td>
			<td>onpg</td>
			<td>是的</td>
			<td>和 #62; 0.99</td>
			<td>4.327 ±1.289</td>
			<td>-1.736 ±0.087</td>
			<td>4.654 ±3.087</td>
			<td>66.37 ±10.75</td>
		</tr>
		<tr>
			<td>er</td>
			<td>CPRG</td>
			<td>不</td>
			<td>= 0.99</td>
			<td>5.673 ±1.764</td>
			<td>-1.914 ±0.052</td>
			<td>3.925 ±1.679</td>
			<td>28.05 ±2.334</td>
		</tr>
		<tr>
			<td>er</td>
			<td>CPRG</td>
			<td>是的</td>
			<td>和 #62; 0.99</td>
			<td>4.412 ±1.142</td>
			<td>-1.894 ±0.051</td>
			<td>2.052 ±1.303</td>
			<td>22.64 ±2.047</td>
		</tr>
	</tbody>
</table>
表3。在图2中, 标准曲线的4参数逻辑回归的模型参数 .两个基质, ONPG 和 CPRG, 测试使用酵母表达 1 2 人类雌激素受体 (ERα或 er) 两个没有和六天的冷藏期后, 17 h 孵化。参数被报告为估计±standard 错误。
附录1。用于计算 LacZ 值的应用程序. 要使用该应用程序, 请首先下载免费的 Java 软件 (如材料表中所述)。然后打开 LacZ 应用程序。与应用程序一起使用的板布局必须与图 1中所示的板布局相同。如果不使用一些取样井, 则保留空井的吸光度读数, 作为吸收数据集的位置持有者, 被粘贴到 LacZ 应用中。 这样可以在应用程序中保留板的空间布局, 并确保介质控制井位于其所需位置。 此外, 如果有空单元格, 则不会执行应用程序。使用键盘命令 "控制 (ctrl) + v" 或 "命令 + v" (根据所使用的计算机平台), 在所有井的 od405读数 (对于 ONPG) 或 od574读数 (对于 CPRG) 进行粘贴。在小时中输入用于生成颜色的检测的孵育时间量 (从步骤5.4 开始, 例如, 0.66667 h 为 40 min)。单击 "下一步"。在步骤5.3 的 OD610读数中粘贴。单击 "提交"。根据所使用的计算机平台, 将显示 LacZ 结果, 并可通过按 "控制 (ctrl) + c" 或 "命令 + c" 复制。<a href="http://ecsource.jove.com/files/ftp_upload/55754/ResearchOp.jar">请单击此处下载此文件.</a>
附录2。用于计算 EEQs 的统计软件选项.EEQs 可以使用三个提供的选项之一进行计算。 使用1的方向。就业选配软件或2。R 代码载于附录2。 R 代码也被转换为3。可在 https://furmanbiology.shinyapps.io/YESapp/上使用的应用程序格式。<a href="http://ecsource.jove.com/files/ftp_upload/55754/Appendix_2_JMP_and_R_directions.docx">请单击此处下载此文件.</a>
附录3。样品收集的数据使用酵母表达 ERα和 CPRG 作为基质.包括对七 E2 标准、车辆和媒体控制以及单个代表性的 PCP 样本, 以及计算出的 LacZ 值, 分别测量 od610和 od574 。LacZ 标准的价值是用来构建一个四参数的标准曲线的逻辑回归模型, 如步骤7A 和 #38; B。用该模型对微井中代表性样品的 EEQs 进行了三项估计。然后这些值被转换为 EEQs 表示为样品的 ng/g (步骤 8.1)。<a href="http://ecsource.jove.com/files/ftp_upload/55754/Appendix_3_example_data_-_alpha_cprg.xlsx">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory at JoVE.com

使用针对本科教学实验室优化的酵母雌激素筛查检测个人护理产品中的雌激素配体 |生物学 |视频

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

本文介绍了一个优化酵母雌激素屏幕的量化配体的个人护理产品 (杆), 结合雌激素受体α (ERα) 和/或β (er)。该方法采用了两种色度基质选择, 一个六天的冷冻孵化, 用于本科课程, 以及数据分析的统计工具。

应用酵母雌激素筛检优选本科教学实验室的个人护理产品中的雌激素配体

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine ...

detecting-estrogenic-ligands-personal-care-products-using-yeast

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13.0K 次观看.  University of the South. 本文介绍了一个优化酵母雌激素屏幕的量化配体的个人护理产品 (杆), 结合雌激素受体α (ERα) 和/或β (er)。该方法采用了两种色度基质选择, 一个六天的冷冻孵化, 用于本科课程, 以及数据分析的统计工具。

视频: Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to &gt;98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.

实时定量PCR使用的Thermo Scientific的Solaris定量PCR检测

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

科研

生物学

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

使用数字免疫荧光显微镜成像在大鼠软脑膜小动脉雌激素受体α

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

定期保养和维护的斑马鱼（<em&gt;斑马鱼</em&gt;）实验室简介

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

反向酵母双杂交系统，以确定哺乳动物核受体残留的互动与配体和/或拮抗剂

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways

Functional complementation assay (FCA) is an in vivo assay that is widely used to elucidate the function/role of genes/enzymes. This technique is very common in biochemistry, genetics and many other disciplines. A comprehensive overview of the technique to supplement the teaching of biochemical pathways pertaining to amino acids, peptidoglycan and the bacterial stringent response is reported in this manuscript. Two cDNAs from the model plant organism Arabidopsis thaliana that are involved in the metabolism of lysine (L,L-diaminopimelate aminotransferase (dapL) and tyrosine aminotransferase (tyrB) involved in the metabolism of tyrosine and phenylalanine are highlighted. In addition, the bacterial peptidoglycan anabolic pathway is highlighted through the analysis of the UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-meso-2,6-diaminopimelate ligase (murE) gene from the bacterium Verrucomicrobium spinosum involved in the cross-linking of peptidoglycan. The bacterial stringent response is also reported through the analysis of the rsh (relA/spoT homolog) bifunctional gene responsible for a hyper-mucoid phenotype in the bacterium Novosphingobium sp. Four examples of FCA are presented. The video will focus on three of them, namely lysine, peptidoglycan and the stringent response.

Functional Complementation Analysis FCA: A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways

The validation of enzymatic activities involved in biochemical pathways can be elucidated using functional complementation analysis (FCA). Described in this manuscript is the FCA assay demonstrating the enzymatic activity of enzymes involved in the metabolism of amino acids, bacterial stringent response and bacterial peptidoglycan biosynthesis.

功能互补分析（FCA）:实验室练习设计和实施，以补充生化途径的教学

Functional complementation assay (FCA) is an in vivo assay that is widely used to elucidate the function/role of genes/enzymes. This technique is very ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯:天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

实验教学中乳糖酶活性的测定

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...