作者想确认朱莉娅 Geminn 在肥大细胞的制备和培养方面的技术援助。这项工作得到了跨区协作研究中心 (SFB) 152 的支持。

所有动物规程根据德国立法指南被执行了对实验室动物的关心和用途 (正式地批准由卡尔斯鲁厄区域理事会)。
1. 经腹腔灌洗的 PMC 分离和培养
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>1</td>
			<td>冰</td>
		</tr>
<tr>
<td>2</td>
			<td>10毫升注射器</td>
		</tr>
<tr>
<td>3</td>
			<td>27克针</td>
		</tr>
<tr>
<td>4</td>
			<td>20克针</td>
		</tr>
<tr>
<td>5</td>
			<td>泡沫塑料块和别针</td>
		</tr>
<tr>
<td>6</td>
			<td>收集管 (50 毫升塑料离心管)</td>
		</tr>
<tr>
<td>7</td>
			<td>70% 乙醇</td>
		</tr>
<tr>
<td>8</td>
			<td>RPMI 介质 (预冷和保持在冰上)</td>
		</tr>
<tr>
<td>9</td>
			<td>PMC 培养基: RPMI 中等 + 20% FCS (胎小牛血清) + 1% 青霉素链霉素溶液 (笔链球菌)</td>
		</tr>
<tr>
<td>10</td>
			<td>Dulbecco 磷酸盐缓冲盐水 DPBS (没有钙2 +和镁2 +)</td>
		</tr>
<tr>
<td>11</td>
			<td>培养烧瓶 (25 厘米)</td>
		</tr>
<tr>
<td>12</td>
			<td>生长因子库存解决方案 (IL-3: 1 微克/ul;超临界: 2.5 微克/毫升)</td>
		</tr>
<tr>
<td>13</td>
			<td>血清吸管 (10 毫升)</td>
		</tr>
<tr>
<td>14</td>
			<td>吸管提示无菌 (20–200µL)</td>
		</tr>
<tr>
<td>15</td>
			<td>Hemocytometer</td>
		</tr>
<tr>
<td>16</td>
			<td>台式离心机</td>
		</tr>
<tr>
<td>17</td>
			<td>剪刀和镊子</td>
		</tr>
<tr>
<td>18</td>
			<td>联合2小鼠室</td>
		</tr>
<tr>
<td>19</td>
			<td>开放式无菌罩</td>
		</tr>
<tr>
<td>20</td>
			<td>封闭式无菌罩</td>
		</tr>
<tr>
<td>21</td>
			<td>细胞孵化器 (37 °c 和 5% CO2)</td>
		</tr>
<tr>
<td>22</td>
			<td>转移吸管 (20–200µL)</td>
		</tr>
</tbody></table>表 1: 步骤1的材料。
<ol><li>
腹腔灌洗分离 PMC
	<ol>
<li>在细胞隔离之前, 准备表 1中列出的材料。</li>
		<li>用8到14周大的雄性小鼠进行 PMC 隔离。弄死一只老鼠, 共2吸入, 并确认死亡的反射损失。用70% 乙醇喷雾鼠标, 用别针 (背侧向下) 将其固定在泡沫块上。使用钝刃剪刀去除鼠标的腹侧皮肤。避免损害腹膜腔。 注意: 我们通常使用这个协议为 C57BL/6N 菌株遗传背景的小鼠。</li>
		<li>使用装有27克针的10毫升注射器, 在腹膜腔中注入7毫升的冰冷 RPMI 培养基和5毫升的空气。在腹膜中小心地推针, 不要穿孔任何器官。使用在附睾脂肪区域的斑点, 以减少器官穿孔的风险。</li>
		<li>注射后, 摇动鼠标1分钟, 将腹膜细胞分离到 RPMI 培养基中。不要太强烈地摇动鼠标, 避免损害内脏, 并用血液污染腹膜腔。</li>
		<li>用新的20克套管将10毫升注射器重新使用。将内脏转移到一侧, 通过倾斜泡沫块, 轻轻地拍打在长凳上, 使中吸更容易从另一侧。插入20克针, 斜角向上, 并从腹部轻轻地和缓慢地吸入液体 (~ 0.5 毫升/秒), 以避免内脏堵塞。收集尽可能多的液体 (通常5–6毫升)。</li>
		<li>从注射器中取出针头, 将收集到的细胞悬浮液转移到冰上的收集管中。如果有明显的血液污染, 就丢弃样品管。</li>
		<li>离心管与细胞悬浮在 ~ 300 x g 5 分钟。在无菌罩下吸上清。对于每只老鼠, 将样品丸组合在4毫升的冷 (4–10°c) PMC 培养基中, 将细胞悬浮液转移到25厘米2培养瓶中。将生长因子 IL-3 和超临界增加到最终浓度 10 ng/毫升和 30 ng/毫升分别。将含有细胞的烧瓶放在孵化器中 (37 摄氏度和 5% CO2), 并孵化为 ~ 48 小时直到下一个过程。</li>
	</ol>
</li>
	<li>
PMC 文化
	<ol>
<li>2天 (隔离后48小时): 中等变化<ol>
<li>要去除上清和非黏附细胞 (红细胞和死细胞), 通过放置一个巴斯德吸管尖端在烧瓶的边缘, 并保持瓶几乎水平, 从烧瓶中吸入培养基。每只鼠标添加4毫升预预热的 PMC 介质。添加生长因子 IL-3 (10 ng/毫升) 和超临界 (30 ng/毫升)。将含有细胞的烧瓶放在孵化器中 (37 摄氏度和 5% CO2)。</li>
		</ol>
</li>
		<li>天 9: 细胞分裂<ol>
<li>将细胞悬浮液从细胞培养瓶中转移到50毫升的塑料离心管中。用10毫升预热 (37 °c) Dulbecco 的磷酸盐缓冲盐水 (DPBS) 清洗烧瓶三次, 用同50毫升塑料离心管中的分离细胞收集细胞悬浮液。用 hemocytometer 计数细胞浓度, 计算细胞总数。</li>
			<li>离心细胞悬浮在 ~ 300 x g 5 分钟, 并吸入上清。恢复预预热的 PMC 培养基 (37 °c) 的颗粒, 以获得细胞浓度 1 x 106细胞/毫升。将细胞悬浮液转移到新的25厘米2培养瓶中。将旧烧瓶与成纤维细胞附着在底部。</li>
			<li>添加生长因子 IL-3 (10 ng/毫升) 和超临界 (30 ng/毫升), 并放置含有细胞在孵化器 (37 °c 和 5% CO2) 的烧瓶。</li>
		</ol>
</li>
		<li>天 12–15: 细胞测量<ol>
<li>如果有任何刺激 FcɛRI 受体的实验计划, 在标准培养基中, 用300毫升的 IgE 抗预处理细胞过夜。继续执行步骤2或步骤3。</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. 临时市政局荧光细胞内游离钙浓度测定
<ol><li>在测量之前, 准备表 2中列出的材料。此外, 准备一个成像设置包括: 倒置荧光显微镜;目的:40 x/1.3 油;Fura 2 过滤器集;CCD 摄像机;励磁光源;图像采集程序;重力馈液应用系统。</li>
</ol><table fo:keep-together.within-page="1"><tbody>
<tr>
<td>1</td>
			<td>PMC (12–15天) 1 x 106细胞/毫升</td>
		</tr>
<tr>
<td>2</td>
			<td>刀豆蛋白 A</td>
		</tr>
<tr>
<td>3</td>
			<td>Fura-2 是</td>
		</tr>
<tr>
<td>4</td>
			<td>Pluronic F-127 20% 解决方案</td>
		</tr>
<tr>
<td>5</td>
			<td>盖滑眼镜轮;ø25毫米;1号</td>
		</tr>
<tr>
<td>6</td>
			<td>dinitrophenol-人血清白蛋白) 库存溶液</td>
		</tr>
<tr>
<td>7</td>
			<td>抗扩散抗体 (IgE) 库存解决方案</td>
		</tr>
<tr>
<td>8</td>
			<td>平底板 (12 井)</td>
		</tr>
<tr>
<td>9</td>
			<td>复合48/80 库存解决方案</td>
		</tr>
<tr>
<td>10</td>
			<td>覆盖井成像室</td>
		</tr>
<tr>
<td>11</td>
			<td>Hemocytometer</td>
		</tr>
<tr>
<td>12</td>
			<td>转移吸管 (20–200µL)</td>
		</tr>
</tbody></table>表 2: 步骤2的材料。
<ol start="2"><li>
Fura-2 成像准备
	<ol>
<li>使用 hemocytometer 执行细胞计数, 并将细胞浓度调整为 1 x 106细胞/毫升。用1毫升的吸管尖轻轻吹打 (3–5倍) 分裂两局殖民地。</li>
		<li>准备一个 ca2 +含 HEPES 缓冲盐溶液 (ca2 +HBSS) 由135毫米氯化钠, 6 毫米氯化钾, 2 毫米 CaCl2, 1.2 毫米氯化镁2, 10 毫米 HEPES 和12毫米葡萄糖组成。用3米氢氧化钠将 pH 值调整为7.4。</li>
		<li>在无菌罩下, 在每25毫米圆盖玻璃上吹打1滴刀豆蛋白溶液 (0.1 毫克/毫升), 准备刀豆蛋白涂覆的幻灯片。把水滴放在盖子上至少1分钟, 然后用吸管除去大部分溶液, 让盖子滑干45分钟. 仔细按刀豆蛋白在橡胶成像室上处理过的盖子滑 (见材料表), 直到他们附上获得显微镜成像室。 注: 聚 l-赖氨酸涂层可作为替代品使用。</li>
		<li>将荧光钙2 +染料 Fura-2 AM (1 毫米) 溶于二甲基亚砜 (亚砜)。存储此解决方案免受光照的侵害。</li>
		<li>稀释 Fura-2 在 Ca2 +HBSS 的库存解决方案, 最终浓度为5µM, 以准备 "加载缓冲区"。补充5µL/毫升 20% pluronic F-127 在亚砜。</li>
		<li>混合500µL 的 "加载缓冲区" 与500µL 的 PMC 细胞悬浮。将混合物注入成像室, 在黑暗中在室温下孵化细胞20–30分钟。</li>
		<li>根据激励协议, 建立具有不同解决方案的重力馈电应用系统。补充注射器1与40毫升的 ca2 +HBSS 解决方案, 注射器2与40毫升的 ca2 +-HBSS 解决方案, 其中包含 100 ng/毫升, 注射器3与40毫升的 Ca2 +-HBSS 解决方案包含50µg/毫升化合物 48/80, 注射器4与40毫升的名义上 ca2 +自由 HBSS 解决方案, 和注射器5与40毫升名义上 ca2 +自由 HBSS 解决方案, 其中包含2µM Thapsigargin。</li>
		<li>准备一个40X 高孔径油浸泡目标, 放置一小滴浸入油。</li>
		<li>在倒置显微镜的舞台上安装应用系统。将成像室与加载的单元格放在应用系统中, 并确保其安全以防止录制过程中的移动。打开传送的光并聚焦细胞。</li>
		<li>使用重力馈的应用系统, 用 Ca2 +HBSS 缓冲器冲洗细胞三次。</li>
		<li>将 Ca2 +-HBSS 中的细胞孵化为5分钟, Fura-2 的酯化反应。</li>
		<li>在开始成像之前再冲洗一次细胞, 清除任何可能泄漏的荧光染料。</li>
	</ol>
</li>
	<li>
细胞成像
	<ol>
<li>在生理采集模式下启动图像采集程序。打开设置窗口, 并调整焦点和最佳捕获时间 (这应该是相同的励磁与340毫微米和380毫微米和通常范围之间 50–150 ms)。 注: 尽量减少 Fura-2 加载单元的曝光量, 以避免染料漂白。</li>
		<li>对引用图片进行图像处理, 并将单元格标记为感兴趣的区域 (ROI)。在不存在单元格的区域中标记最后的 ROI, 并将其定义为背景。</li>
		<li>完成安装, 并开始测量的 5 s 的购置率。 注: 细胞将交替照明与激发光340毫微米和 380 nm, 并发射荧光 (> 510 nm) 将收集使用 CCD 相机。荧光信号 F340 纳米和 F380 nm 的图像以及比值 (F340 nm/F380 nm) 将显示在三窗口中。个体 ROIs 荧光强度平均值的时间过程图也将持续显示。</li>
		<li>根据实验设计, 在适当的周期编号中添加解决方案:<ol>
<li>为了测量抗原诱导海拔的 [Ca2 +]i, 如图 2A, 2B, 在循环20后应用注射器2解决方案, 并停止记录后, 周期150。</li>
			<li>要测量 [Ca2 +]i的复合48/80 诱导海拔, 如图 2C所示, 在循环20后应用注射器3解决方案, 并在周期150之后停止录制。</li>
			<li>要测量由 SOCE 诱发的 [Ca2 +] 的海拔, 如图 2D所示, 在循环10后应用注射器4溶液, 在循环5后应用注射器70溶液, 在循环4后应用注射器120溶液,在循环150后应用注射器1解决方案, 并在220周期后停止录制。</li>
		</ol>
</li>
		<li>完成测量后, 将结果转换为包含和不带背景校正的励磁波长的测量强度的比值表, 以及每个 ROI 的比率值和没有背景校正的比值。和每个测量时间点。在采集程序中, 依次按下按钮: "启动刀"、"全部标记"、"全部转换"、"生理学测量"、"确定"、"确定"。将文件保存为表和图像堆栈以进行离线分析。</li>
	</ol>
</li>
</ol>3. hexosaminidase 释放量的测试
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>1</td>
			<td>PMC (12–15天) 1 x 106细胞/毫升</td>
		</tr>
<tr>
<td>2</td>
			<td>抗扩散抗体 (IgE) 库存解决方案</td>
		</tr>
<tr>
<td>3</td>
			<td>白蛋白库存解决方案</td>
		</tr>
<tr>
<td>4</td>
			<td>复合48/80 库存解决方案</td>
		</tr>
<tr>
<td>5</td>
			<td>Ionomycin</td>
		</tr>
<tr>
<td>6</td>
			<td>96-井板, v 形底部</td>
		</tr>
<tr>
<td>7</td>
			<td>96-井板, 平底</td>
		</tr>
<tr>
<td>8</td>
			<td>塑料离心管 (15 毫升)</td>
		</tr>
<tr>
<td>9</td>
			<td>血清吸管</td>
		</tr>
<tr>
<td>10</td>
			<td>细胞孵化器 (37 °c 和 5% CO2)</td>
		</tr>
<tr>
<td>11</td>
			<td>台式离心机</td>
		</tr>
<tr>
<td>12</td>
			<td>用于光学密度测量的微量滴定板读取器</td>
		</tr>
<tr>
<td>13</td>
			<td>多通道吸管 (20–200 ul)</td>
		</tr>
<tr>
<td>14</td>
			<td>Hemocytometer</td>
		</tr>
</tbody></table>表 3: 步骤3的材料。
<ol><li>在测量之前, 准备表 3中列出的材料。 注: β-hexosaminidase 释放从 MCs 后, 他们的刺激水解对硝基苯基-乙酰氨基葡萄糖 (pNAG) 成对硝基酚和 n-乙酰氨基葡萄糖。样品中β-hexosaminidase 的数量与将形成的对硝基酚的数量成正比。在 "停止溶液" 的高 pH 环境下, 对硝基酚作为完全 deprotonated 的 nitrophenolat 存在, 可通过其光吸收率在 405 nm 上检测到。</li>
	<li>准备 Tyrode 的溶液, 含130毫米氯化钠, 5 毫米氯化钾, 1.4 毫米 CaCl2, 1 毫米氯化镁2, 10 毫米 HEPES, 5.6 毫米葡萄糖和 BSA 0.1%。用3米氢氧化钠将 pH 值调整为7.4。</li>
	<li>在 Tyrode 的溶液中加入1% 体积的海卫 X-100 来制备裂解液。</li>
	<li>制备由 200 mM 甘氨酸组成的止水溶液, 并用氢氧化钠调整 pH 值为10.7。</li>
	<li>pNAG 溶液 (4-硝基苯基 2-acetamido 2-脱氧β D-glucopyranoside) 溶液通过溶解 pNAG 在 0.4 M 柠檬酸到最后浓度4毫米。</li>
	<li>计算化验所需的细胞量 (重复执行化验)。每井使用20万个临时市政局。使用2口井作为负控制 (Tyrode 的解决方案, 如 secretagogues 或复合 48/80) 和2井作为正控制 (Tyrode 的解决与10µM Ionomycin) 为每个基因型。</li>
	<li>将这些细胞转换成15毫升的塑料离心管, 用 Tyrode 的溶液调整体积到15毫升, 离心机在 200 x g处进行4分钟. 用巴斯德吸管取出上清液, 并并用重悬 Tyrode 溶液中的细胞为 2 x 106细胞毫升.</li>
	<li>将细胞悬浮液的100µL 转移到一个96井的 V 型井底井板上。将刺激或控制解决方案的25µL 添加到每个井中 (根据图 3A所示的吹打方案)。孵化细胞45分钟在37°c 和 5% CO2。</li>
	<li>把96井板放在冰上5分钟, 停止反应。然后, 离心板在4°c 为4分钟在 120 x g。用多通道吸管将上清的120µL 转移到平底96井板上, 放在冰上。小心避免接触细胞小球, 但完全吸入上清。</li>
	<li>将125µL 的溶解缓冲液添加到细胞颗粒中。在室温下孵化细胞颗粒5分钟, 并在孵化后并用重悬, 重复吹打 (约5次)。</li>
	<li>吸管25µL 的 pNAG 溶液 (4 毫米) 在所需的井中的一个新的平底96井板。添加25µL 从每个上清到准备好的 pNAG 溶液以及每个细胞裂解液的25µL。</li>
	<li>在37摄氏度孵育1小时的反应批次。孵化后, 吸管150µL 的停止溶液, 使各井停止反应。</li>
	<li>使用 405 nm 的双波长设置与参考 630 nm 进行自动背景减法分析, 微板块读取器。在采集程序中, 勾选框 "参考", 并在 "吸光度" 程序元素菜单中, 从下拉列表中选择 "630 nm"。如果样品的光学密度太高, 在 remeasuring 之前准备适当稀释探针。</li>
	<li>根据以下公式计算β hexosaminidase 释放的百分比: <img alt="Equation 1" src="/files/ftp_upload/57222/57222eq1.jpg"> 如果释放 [%] 是特定β-hexosaminidase 释放的百分比, [上清] 是对上清的背景校正吸收, 而 [裂解] 是对相应细胞裂解的背景校正吸收。</li>
</ol>

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作者没有什么可透露的。

mc 模型可以成功地用于研究 mc 脱粒, 趋化性, 黏附性, 以及阐明细胞内信号传导通路参与 mc 激活。一些研究人员使用人体 mcs 模型, 如永生化的线 (LAD2 和 HMC1) 或由脐血祖细胞 (CD133+) 和外周血祖细胞 (CD34+) 所衍生的人 mcs。其他人更喜欢啮齿动物 MC 模型, 如永生化 (大鼠嗜碱性颗粒白血病 RBL-2H3 MC 线) 或初级培养 (小鼠骨髓源性 MCs BMMCs, 局) 模型。小鼠原发性 MCs 可用于分析多种 "敲出" 老鼠模型中的 MC 功能, 这是探索特定基因和编码蛋白生理功能的黄金标准方法。由于缺乏足够的选择性和有效性的药理工具, 这一方法对于研究 MC 激活18的信号传导机制特别有价值。小鼠局是结缔组织表型, 呈现成熟的形态学和高粒度。它们不仅对 FcεRI 类似 BMMCs 的抗原交联反应良好, 而且还由几种附加受体的激动剂激活, 如内皮素1或复方48。然而, 与 BMMCs 相比, 临时市政局的收益率通常明显较低, 通常不足以执行西方污点或其他需要大量细胞的技术。本文介绍了几种分离纯化的方法。例如, Percoll 梯度离心被报告产生高纯度的细胞19;然而, 用这种技术获得的细胞数量通常相对较低。在这里描述的协议中, 最关键的步骤是细胞悬浮从腹腔腔的渴望。避免样品的血液污染, 最大限度地减少培养基的含量, 对于获得高纯度的局数是至关重要的。与一些小鼠菌株, 一个最大的 PMC 数只获得了缩短 (11–12天) 培养期。离开这些细胞在文化条件为更长的时间仅导致细胞数字的减少。
在本研究中, 我们描述了一个简单的协议, 适合隔离成熟的 MCs, 使其能够获得一个高度纯净的 MC 人口从小鼠腹膜腔。所获得的细胞具有高同质性, 表现出典型的 MC 特征, 如特定标记受体 (试剂盒和 FcεRI) 的表达。这些细胞清楚地展示 MC 特征到 degranulate 并且增加他们的 [Ca2 +]i为响应 FcɛRI 和 MRGPR 激活。
MC 信号是紧密相关的 [ca2 +]i水平确定由 ca2 +释放从胞内存储和 ca2 +进入通过等离子膜通道。[Ca2 +]我的海拔激活一个复杂的细胞内信号通路, 触发 MC 脱粒。与其他非易激动细胞一样, MCs 中的主要 ca2 +流入通路是由 ca2 +存储的耗尽所激活的 SOCE。识别信号通路所涉及的通道对于理解 MC 函数的调节至关重要。对于 SOCE 通路分子成分的鉴定, 采用荧光钙2 +指标的 microfluorometric 技术和 "膜片钳" 电生理方法起到了突出的作用。但是, [ca2 +]i级的提升是 Ca2 +释放和 SOCE 的总和。为了区分这两个阶段的 ca2 +动员, 所谓的 "Ca2 +重新添加" 协议 (审查见鸟类等人 200820) 以前开发。在本协议中, 外部盐解决方案从一个包含正常 [ca2 +]o (.在这些条件下, 细胞被 thapsigargin 到空的 Ca2 +商店, 从而完全激活 SOCE。在没有胞外 ca2 +, thapsigargin 治疗唤起瞬态 [ca2 +]i海拔, 反映出 ca2 +离子从胞内 ca2 +存储。再添加胞外 Ca2 +导致增加 [Ca2 +]我代表 SOCE。本文介绍了该方法在 MCs 中 SOCE 特性分析中的成功应用。为了监视 [Ca2 +]i变化, Fura-2 被用作钙指示器。在它的乙酰甲基形式, Fura-2 可以很容易地被装载在临时市政局。使用这种比例染料不仅能比较 [ca2 +]i海拔的振幅, 而且还可以对比基底 [ca2 +]i水平的差异, 这对于分析野生类型/挖空尤为重要。细胞。与基因编码的荧光染料相比, Fura-2 的主要缺点之一是其在不同细胞器中的高积累, 从而对细胞质荧光的污染。
Fura-2 成像需要双励磁技术。通常, 它在技术上比双重发射技术更容易使用, 它不仅要求触发光源的使用, 而且还需要在发射光路径中加入过滤轮或图像分配器。所描述的协议可用于其他非易激动细胞的 Ca2 +成像。
MCs 的一个重要特点是其高含量的电子致密分泌颗粒, 充满了大量的调解人和免疫调节物质。MC 活化导致了预制颗粒化合物的快速释放。脱粒体外分析的几种方法, 包括核素血清素的检测, 抗体 (ELISA) 的组胺检测, 以及利用电生理 "膜片钳" 细胞电容测量。本文介绍了在体外释放β-hexosaminidase 的多井比色检测法的实际应用。本试验的基础是β-hexosaminidase 水解n-乙酰基-乙酰-d--d--hexosaminides21中氨基己糖残留物的能力。β-hexosaminidase 与组胺共用, 但也与其他调解人如蛋白酶 D 或 TNF, 因此可以作为一个关联的脱粒从多种类型的分泌囊泡在 MCs12,22。
在所述技术中, 以不同 secretagogues 的37°c 刺激了局地, 然后通过与β hexosaminidase 基质的反应, 对上清液和β hexosaminidase 含量的颗粒进行了估算。作为基体, 4-硝基苯基 n-乙酰基β glucosaminide 被使用。水解成 n-乙酰氨基葡萄糖和对硝基酚. 对硝基苯酚的数量 colorimetrically 量化的光吸收率在 405 nm。在ß-hexosaminidase 释放试验的一些修改中, 4-methylumbelliferil n-乙酰氨基葡萄糖被用作基质。酶水解后, 该物质产生 fluorometrically 可检测化合物。我们将脱粒的范围表示为发布的百分比, 并将其规范化为总内容。这使我们能够避免不同细胞数和它们在各种细胞制剂之间的颗粒含量所引入的变异。我们没有减去一个自发释放 (在没有 MCs secretagogues 的情况下观察) 从净刺激脱粒, 因为自发释放被单独报告, 因为它的重要性作为一个指标的 MC 生存能力。为了避免测定结果的高变异性, 在被刺激的细胞离心后非常仔细地分离上清液和颗粒是很重要的。在执行比色测量之前, 避免在多井板中形成任何气泡也是必不可少的。所述方法的一个重要限制是, 结果表示不作为绝对值, 但作为释放的中介的百分比。
与直接检测释放的血清素或组胺的方法相比, 报告的化验法更符合成本效益, 不需要专门的设备。此外, 所述技术具有高度重现性, 不需要在放射性实验室进行, 也不需要购买昂贵的抗体。因此, 它可能是一个很好的替代更昂贵和困难的技术。

MCs 在先天和后天免疫应答中起着突出的作用。具体地说, MCs 参与杀害病原体, 如细菌和寄生虫, 并降低潜在的有毒内源性肽或毒液成分 (审查见加利等20081)。MCs 在先天和适应性免疫中的生理作用是激烈争论的主题。因此, 在不同的 MC 缺陷小鼠模型的研究中, 大量的数据差异需要对2过敏后的 MCs 免疫功能进行系统的重新评估。成熟的 MCs 大多是局部的组织和器官, 如皮肤, 肺和肠道, 通常只在少量的血液中发现。MCs 来源于造血前体, 如 MC 祖细胞, 在几乎所有血管化组织的微环境中完成它们的分化和成熟1。T 细胞衍生因子白细胞介素 (IL)-3 有选择地促进小鼠 MCs 的多能种群的生存能力, 增殖和分化从他们的造血祖父母3。干细胞因子 (超临界) 是由组织中的结构细胞产生的, 在 MC 的发展、生存、迁移和功能4中起着至关重要的作用。单个 MCs 的性质可能因其合成和贮存各种蛋白酶或软骨的能力而异。在小鼠中, 根据肝素和蛋白酶5的解剖定位、形态学和含量, 将所谓的结缔组织型 mcs 与黏膜 mcs 区别开来。
在 MCs 中, 增加游离胞浆 Ca2 + ([Ca2 +]i) 是脱粒和生产 eicosanoids, 以及细胞因子的合成和转录因子的活化反应的抗原和各种 secretagogues6。这些刺激的主要下游目标是磷脂酶 C, 水解磷酸肌醇 45-bisphosphate 到甘油 (达格·哈马舍尔德) 和肌醇 14, 5-trisphosphate。IP3 激活蛋白激酶 C, 并从内质网中释放出钙2 +的离子。这些商店的枯竭激活了 ca2 +通过等离子膜 ca2 +通道流入, 导致储存操作钙进入 (SOCE)。这一过程是通过与 Ca2 +传感器, 基质相互作用 molecule-1 (Stim1) 的互动, 在内质网与 Orai17以及通过活化的瞬态受体电位规范 (TRPC) 通道蛋白质 (复习见 Freichel等。20148) 在血浆膜。
为了研究这些通道的生理作用, 通常使用几种药理学 (通道阻滞剂的应用) 或遗传方法。在后一种情况下, 抑制蛋白质表达是通过靶向 mRNA (击倒) 或基因组 DNA 编辑与全球或组织特定的9删除一个外显子编码一个孔形成亚单位的通道 (挖空)。对这些通道有足够特异性的拦截剂的供应是有限的。此外, 被击倒的方法需要仔细控制它的有效性使用, e., 西方污点分析, 并由于缺乏特定的抗体为靶向的渠道蛋白在许多情况下。因此, 剔除鼠标模型的使用仍然被认为是这种分析的黄金标准。对 MC 功能进行调查的一种优选的体外模型是培养可被隔离为完全成熟的种群 (相对于体外, e., 骨髓细胞的不同的 MCs)10.与骨髓源性 MCs (BMMCs) 相比, 它也通常用于研究 MC 功能的体外, FcεRI 和β hexosaminidase 释放的刺激分别增加8倍和100倍。在本文中, 我们描述了一种分离和培养小鼠的方法, 它允许在2周后获得大量的纯结缔组织类型的 MCs, 足以进一步分析。
尽管最近在开发和应用基因编码钙指标方面取得了进展, 但它们的使用仍受限于向目标细胞提供基因的困难, 特别是在诸如初级培养的 MCs 等高度专门化的细胞中。此外, 这组荧光指示器为 [2 +]i测量仍然缺乏比例染料以一个高动态范围。因此, 比例 [Ca2 +]i测量的首选方法仍然是使用荧光染料 "Fura-2"11。
目前, 对 MC 活化和脱粒的评价最常用的方法是β-hexosaminidase 活性的测量。β-hexosaminidase, 过敏性调解人, 是 MC 颗粒成分之一, 与组胺的联合释放, 从 MCs12的恒定比例。β-hexosaminidase 可以通过其与特定基质的反应而容易而准确地测量, 从而产生可测量的 colorigenic 产品的数量, 在微板块比色法中很容易检测到。本文介绍了该技术在脱粒分析中对各种刺激反应的应用。
高亲和 plasmalemmal IgE 受体 (FcɛRI) 的聚集在 MCs 中激活一个多功能的细胞内信号通路, 导致分泌颗粒含量在周围细胞外空间释放。除了特定的抗原, 许多其他刺激可以激活 MCs 释放多种免疫调节介质, 如补充 anaphylatoxins (e., C3a 和 C5a)13, 收缩肽内皮素 1 (ET1)14, 以及许多阳离子物质和药物引起的 pseudoallergic 反应 (e., icatibant)15通过绑定到 MRGPRX216。MRGPR 诱导的 MCs 脱粒细胞内信号通路与 FcɛRI 介导的细胞内信号传递的特征相比较差;这些途径只开始密集地被研究在最近几年17在受体识别16以后。在很大程度上, 涉及钙进入 MRGPRX2 刺激的等离子膜离子通道仍有待了解。因此, 本文还重点研究了 MRGPR 激动剂刺激的 MCs 细胞内钙信号和脱粒。

<table><tbody><tr><td>RPMI Medium</td><td>Thermo Scientific</td><td>21875034</td><td></td></tr><tr><td>DPBS</td><td>Sigma-Aldrich</td><td>14190144</td><td></td></tr><tr><td>FCS</td><td>Thermo Scientific</td><td>R92157</td><td></td></tr><tr><td>IL-3</td><td>R&amp;amp;D Systems</td><td>403-ML</td><td></td></tr><tr><td>SCF</td><td>Thermo Scientific</td><td>PMC2115</td><td></td></tr><tr><td>Concanavalin A</td><td>Sigma-Aldrich</td><td>C2272</td><td></td></tr><tr><td>Fura-2 AM&amp;nbsp;</td><td>Thermo Fischer Scientific</td><td>F1221</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma-Aldrich</td><td>P2443</td><td></td></tr><tr><td>DNP-HSA&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A6661</td><td></td></tr><tr><td>Anti-DNP-Antibody&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D-8406</td><td></td></tr><tr><td>Penstrep</td><td>Thermo Scientific</td><td>15140122</td><td></td></tr><tr><td>Compound 48/80&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C2313</td><td></td></tr><tr><td>4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>4-Nitrophenyl 2-acetamido-2-deoxy-&amp;beta;-D-glucopyranoside</td><td>Sigma-Aldrich</td><td>N9376</td><td></td></tr><tr><td>CoverWell Imaging Chamber</td><td>Sigma-Aldrich</td><td>GBL635051</td><td></td></tr><tr><td>96-Well plate, v-shaped bottom&amp;nbsp;</td><td>Corning</td><td>3896</td><td></td></tr><tr><td>96-Well plates, flat bottom</td><td>Greiner Bio-One</td><td>655180</td><td></td></tr><tr><td>Microtiter plate reader with &quot;i-control&quot; software&amp;nbsp;</td><td>Tecan</td><td>Nano Quant, infinite M200 Pro</td><td></td></tr><tr><td>Flat bottom plate&amp;nbsp;</td><td>Greiner Bio-One</td><td>655180</td><td></td></tr><tr><td>Ionomycin</td><td>Sigma-Aldrich</td><td>I9657</td><td></td></tr><tr><td>50 mL Plastic Centrifuge Tubes</td><td>Sarstedt</td><td>62.547.254&amp;nbsp;</td><td></td></tr><tr><td>Culture Flasks (25 cm)</td><td>Greiner Bio-One</td><td>69160</td><td></td></tr><tr><td>Cover slips glasses round; &amp;oslash; 25 mm; No. 1</td><td>Menzel</td><td>CB00250RA1</td><td></td></tr><tr><td>Hemocytometer</td><td>VWR</td><td>631-0696</td><td></td></tr><tr><td>Inverted Microscope</td><td>Zeiss</td><td>Observer Z1</td><td></td></tr><tr><td>Objectiv Fluar 40x/1.3 Oil</td><td>Zeiss</td><td>440260-9900</td><td></td></tr><tr><td>HC Filter Set Fura 2&amp;nbsp;</td><td>AHF</td><td>H76-521</td><td></td></tr><tr><td>CCD Camera</td><td>Zeiss</td><td>AxioCam MRm&amp;nbsp;</td><td></td></tr><tr><td>Monochromatic Light Source</td><td>Sutter Instruments</td><td>Lambda DG-4</td><td></td></tr><tr><td>Imaging Acquisition Program</td><td>Zeiss</td><td>AxioVision 4.8.2&amp;nbsp;</td><td></td></tr><tr><td>Gravity fed solution application system</td><td>Custom Made</td><td>4-channel</td><td></td></tr><tr><td>15 mL Plastic Centrifuge Tubes</td><td>Sarstedt</td><td>62,554,502</td><td></td></tr><tr><td>Bench Centrifuge</td><td>Heraeus</td><td>Megafuge 1.0R</td><td></td></tr></tbody></table>

isolation of peritoneum-derived mast cells and their functional characterization with ca2+-imaging and degranulation assays

肥大细胞 (MCs) 作为免疫系统的一部分, 在保护宿主抵抗几种病原体和引发过敏性免疫应答方面起着关键作用。通过将表面 IgE 与高亲和 IgE 受体 (FcεRI) 的交联, 以及通过其他几种受体的刺激, 激活 MCs, 启动了游离胞内 Ca2 +级 ([Ca2 +]i) 的上升。这促进了炎症和过敏介质的释放。这些信号通路中所涉及的分子成分的识别对于理解 MC 函数的调节是至关重要的。本文介绍了一种通过腹腔灌洗和腹腔 MCs 培养分离小鼠结缔组织型 mcs 的协议。采用这种方法对各种击倒小鼠模型的 MCs 进行培养, 是鉴别 MC 信号通路所涉及蛋白质的一种有用方法。此外, 我们还描述了一个单细胞 Fura-2 成像协议, 作为一个重要的技术, 以量化的 Ca2 +信号在 MCs. 基于荧光的监测 [ca2 +]我是一个可靠和常用的方法来研究 Ca2 +信号事件, 包括存储操作的钙进入, 这是至关重要的 MC 激活。对于 MC 脱粒的分析, 我们描述了β hexosaminidase 释放试验。将β-hexosaminidase 释放到培养基中的量被认为是 MCs 中描述的所有三种不同分泌亚群的脱粒标记. β-hexosaminidase 可以很容易地被量化的反应与 colorigenic 基板在微量滴定板比色法。这种高度可重复的技术具有成本效益, 不需要专门的设备。总体而言, 所提供的协议表明了一个高产量的 mcs 表达典型的 MC 表面标记, 显示典型的形态和表型特征的 mcs, 并表明高重现性反应 secretagogues 在 Ca2 +-影像学和脱粒化验。

在无菌湿润条件下, 经腹腔灌洗并进一步培养 RPMI 培养基中的3种野生小鼠 (C57BL/6N), 在生长因子 (IL-3 10 ng/毫升和 30 ng/毫升) 的情况下, 对其进行了进一步养殖。37°c 和 5% CO2。媒介在天2和9被改变了在细胞隔离以后。用透射光 DIC 成像 (见图 1A, B) 对细胞形态学进行分析。细胞的对比度和粒度表现出良好的细胞活力。在这些条件下培养2周后, 获得了 1.5 x 107细胞的均匀种群。用 FITC 和抗 c-试剂盒抗体结合 pe 结合抗 IgE 抗体的细胞进行流式细胞分析, 揭示了98.6% 的 FITC 阳性和99.8% 阳性细胞 (图 1c)。98.5% 的培养细胞为 FITC 和 PE 的双阳性 (图 1C), 显示成熟 MCs 的典型特征。
为了进一步的功能分析获得的细胞, 荧光 [Ca2 +]我的测量使用 Fura-2 荧光指示器进行。Fura-2 被装载的细胞交替地被照亮了与340毫微米和380毫微米励磁光每 5 s 和荧光 > 510 毫微米使用 CCD 照相机登记。荧光 (F340/F380) 的比值一直被监测。应用 100 ng/毫升对 PMC 孵化一夜间与 300 ng/毫升的 IgE 反扩散诱发了显着海拔的 [Ca2 +]i水平, 这慢慢腐烂到它的一半的最大数分钟 (图 2a, B)。细胞反应的变异性相对较低 (图 2A, B)。MRGPRB2 受体50µg/毫升的复合48/80 与同一时间协议也大大增加 [Ca2 +]i在 Fura-2-loaded 局, 具有非常相似的平均幅度的响应 (图 2C)。在对 SOCE 的分析中, 采用了 "再添加" 协议: 测量开始后的10个循环, 外部 Ca2 +被移除, 在周期70–120中, Thapsigargin (2 µM) 是内质网 atp 酶抑制剂, 是适用于胞内 Ca2 +存储的耗尽和 SOCE 的最大活化。瞬态 [Ca2 +]i高程反映了 ca2 +释放从胞内 ca2 +存储在名义上 ca2 +免费浴缸解决方案 (图 2D)。在150周期后将外部 Ca2 +浓度恢复到2毫米后, 由于 ca2 +通过 SOCE 通道的涌入 (图 2D), 导致了 [ca2 +]i级的显著增加。ca2 +响应的此组件在存在10µM 葛兰素 7975A (称为裂纹阻滞剂) 时强烈抑制, 而 ca2 +从胞内 Ca2 +存储区的释放保持不变 (图 2D).
在下一步, 我们测试了独立的脱粒在交联 FcɛRI 高亲和受体或刺激 MRGPRB2 受体的能力。为此, 进行了ß hexosaminidase 释放试验。单元格被添加到 V 底96井板 (2 x 105细胞/井), 并根据图 3a所示的方案进行刺激, 45 摄氏度为37分钟。该板为离心, 清除后上清, 颗粒裂解。在37摄氏度的1小时孵化过程中, 通过与 pNAG 的反应, 分析了单个上清液和球团裂解物的ß hexosaminidase 含量。colorimetrically 检测到反应产物的数量, 并计算出释放的ß-hexosaminidase 的百分比 (见图 3B)。自发释放非常低 (1%), 而对强脱粒刺激的反应, 如10µM Ionomycin 和50µg/毫升的复合48/80 分别为40% 和60%。对脱粒的反应是剂量依赖于10–300的浓度范围。这些数据在一起, 清楚地表明孤立的两局的良好的功能状态。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57222/57222fig1.jpg"> 图 1: 采用 DIC 显微术和流式细胞分析法对原代培养的小鼠野生型局进行了表征.(A、 B)使用 20X plasDIC 目标的代表性图像获得 25 cm2塑料烧瓶1小时后腹腔灌洗细胞隔离 (A) 和9天的培养 (B)。右下角的缩放条形图表示100µm. (C) 根据上述协议培养的12天的经体流式细胞仪分析与异硫氰酸荧光素 (FITC) 和抗 c-试剂盒联合染色抗 IgE 抗体。与藻红蛋白 (PE) 共轭的抗体。细胞密度图分为四象限: Q1, PE 高于自体荧光水平/FITC 低于自体荧光水平;Q2, 高于自体荧光水平/FITC 以上的 PE;Q3, 低于自体荧光水平/FITC 以下的 PE;Q4, 低于自体荧光水平/FITC 以上的 PE。<a href="/files/ftp_upload/57222/57222fig1large.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/57222/57222fig2.jpg"> 图 2:记录 [Ca2 +]我在 Fura-2 被从野生型小鼠中分离出来。[Ca2 +]我提出荧光 F340/F380 比.(A) (100 ng/毫升) 刺激的个别局 (n = 38) 的 [Ca2 +]i时间路线的代表性痕迹。(B) 第二次 (100)-诱发的 (Ca2 +]i海拔的时间过程。每个数据点都表示从面板 A 中所示的38个单独单元格中获得的平均值值。这些临时市政局在一夜之间进行了预处理, 300 毫升的 IgE 反扩散。(C) 在临时市政局 (n = 60), 复合48/80 诱导的 [Ca2 +]i海拔的平均值的时间过程。(D) [ca2 +] 的平均值的时间过程在胞内 ca2 +储存损耗期间, 在名义上 ca2 +免费解决方案中应用2µM Thapsigargin (Tg) 时发生变化, 其次是商店操作的钙汇集以后再加法2毫米 Ca2 +在巴恩解决方案在控制小组 (n = 44) 临时市政 (黑正方形) 和在10µM 的存在葛兰素7975A 在浴解答 (蓝色正方形) (n = 41)。在 B、C 和 D 中, 误差条显示平均值的标准误差。<a href="/files/ftp_upload/57222/57222fig2large.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/57222/57222fig3.jpg"> 图 3:与野生型小鼠分离的 hexosaminidase 释放比色多井测量.(A) 96 井板吹打的刺激方案, 用于执行β-hexosaminidase 试验;结果显示在 B 组中. "斯邦坦" 对应于 Tyrode 解决方案的吹打, 而不添加任何 secretagogues;IM = Ionomycin;Cp 48/80 = 复合48/80。(B) 条形图, 说明在基础条件下 (性自然) 的ß-hexosaminidase 释放百分比, 在 Ionomycin (IM)、dinitrophenol-人血清白蛋白 () 和复方 48/80 (Cp 48/80) 的刺激下, 具有指示的浓度。该化验以重复进行; 及误差线显示平均值的标准误差。<a href="/files/ftp_upload/57222/57222fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays at JoVE.com

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

在这里, 我们提出了一个隔离和培养小鼠腹膜肥大细胞的协议。我们还描述了两个协议的功能表征: 一个荧光成像的细胞内游离 Ca2 +浓度和脱粒检测的基础上的色度量化的释放β-hexosaminidase。

腹膜源性肥大细胞的分离及其功能特征的 Ca2 +成像和脱粒测定

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...

isolation-peritoneum-derived-mast-cells-their-functional

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

12.2K 次观看.  Ruprecht-Karls Heidelberg University. 在这里, 我们提出了一个隔离和培养小鼠腹膜肥大细胞的协议。我们还描述了两个协议的功能表征: 一个荧光成像的细胞内游离 Ca2 +浓度和脱粒检测的基础上的色度量化的释放β-hexosaminidase。

视频: Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

心脏功能免疫细胞的分离

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

科研

免疫与感染

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

调查肥大细胞分泌颗粒;从合成到胞吐作用

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such as skin, airways and gastrointestinal tract. Best known for their detrimental role in IgE-dependent allergic reactions, MCs have also emerged as important players in host defense against venom and invading bacteria and parasites. MC phenotype and function can be influenced by microenvironmental factors that may differ according to anatomic location and/or based on the type or stage of development of immune responses. For this reason, we and others have favored in vivo approaches over in vitro methods to gain insight into MC functions. Here, we describe methods for the generation of mouse bone marrow-derived cultured MCs (BMCMCs), their adoptive transfer into genetically MC-deficient mice, and the analysis of the numbers and distribution of adoptively transferred MCs at different anatomical sites. This method, named the &#8216;mast cell knock-in&#8217; approach, has been extensively used over the past 30 years to assess the functions of MCs and MC-derived products in vivo. We discuss the advantages and limitations of this method, in light of alternative approaches that have been developed in recent years.

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

We describe a method for the generation of in vitro derived mast cells, their engraftment into mast cell-deficient mice, and the analysis of the phenotype, numbers and distribution of engrafted mast cells at different anatomical sites. This protocol can be used to assess the functions of mast cells in vivo.

使用"桅杆细胞敲击"小鼠分析体内桅杆细胞的功能

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such ...

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

的Perigenital敏感性和前列腺肥大细胞活化新生母婴分离的小鼠模型评估

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

医学

An Ex vivo Mast Cell Degranulation Assay using Crude Peritoneal Exudate Cells and Natural Antigen Stimulation

Mast cell stabilizers are an essential part of allergy medication. Passive systemic anaphylaxis (PSA) is an animal assay widely used for investigating the effect of a pharmacological agent of interest on mast cells in vivo. As the anaphylactic symptoms are primarily attributed to exocytosis of the granules from mast cells, it is conceived that the agent to cause amelioration of the symptoms has a mast cell stabilizing activity. Despite the fact, it is prudent to confirm the activity by directly demonstrating the decline in the functional activity of mast cells following its treatment. In vitro degranulation assays using an immortalized mast cell line or cultured primary mast cells are routinely employed to that end. The results from the in vitro and in vivo assays may not always be akin to each other; however, as treatment conditions (e.g., treatment dose, time, surrounding environments) for the in vitro assays are often distinct from those for the in vivo assay such as PSA. In pursuit of an in vitro (or ex vivo) assay to reflect more closely the effect of a pharmacological agent on mast cells in vivo, we devised the ex vivo mast cell degranulation assay in which crude peritoneal exudate cells (PECs) isolated from the mice, treated with the agent and administered anti-dinitrophenol (DNP) IgE, were incubated directly with DNP on a carrier protein. It turned out that the assay was not only useful in validating the mast cell stabilizing activity of a pharmacological agent indicated by the in vivo assay but also practical and highly reproducible.

We have established an ex vivo mast cell degranulation assay carried out by incubating crude peritoneal exudate cells isolated from the mice, treated with a pharmacological agent of interest and administered anti-dinitrophenol (DNP) IgE beforehand, with DNP on a carrier protein.

Mast cell stabilizers are an essential part of allergy medication. Passive systemic anaphylaxis (PSA) is an animal assay widely used for investigating the ...

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new therapeutics. Mas related G-protein receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, directs the general immune response. Numerous ligands for MRGPRX2 have been identified and include endogenous peptides like PAMPs, defensins, LL-37 and other protein fragments (i.e., degraded albumin). Further identification of MRGPRX2 specific ligands requires the screening of a large number of peptides (i.e., peptide library); however, mast cells are difficult and expensive to maintain in vitro and, therefore, not economical to use for screening large numbers of molecules. The present paper demonstrates a method to design, develop, and screen a library of small peptide molecules using MRGPRX2 expressing HEK cells. This cell line is relatively easy and inexpensive to maintain and can be used for in vitro high-throughput analysis. A calcium sensitive Fura-2 fluorescent dye to mark intracellular calcium flux upon activation was used to monitor the activation. The ratio of fluorescence intensity of Fura-2 at 510 nm against excitation wavelengths of 340 and 380 nm was used to calculate calcium concentration. The peptide library used to verify this system was based on the endogenous proadrenomedullin N-terminal 12 (PAMP-12) secretagogue, which is known to bind MRGPRX2 with high specificity and affinity. Subsequent peptides were generated through amino acid truncation and alanine scanning techniques applied to PAMP-12. The method described here is simple and inexpensive yet robust for screening a large library of compounds to identify binding domains and other important parameters that play an important role in receptor activation.

Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors.

使用工程 HEK 细胞筛选激活 MRGPRX2 的肽

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

完整、全小鼠乳腺的分离及细胞外基质表达及腺体形态学分析

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

生物学

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

成年小鼠泪液和下颌腺肌上皮细胞的分离

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Mast Cell Degranulation Assay to Study the Effect of a Target Chemical

Source: Tsvilovskyy, V., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57222">Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays.</a>&#160;J. Vis. Exp. (2018).
This video demonstrates a colorimetric assay to determine the degranulation activity of mast cells. The enzyme released by the mast cells hydrolyzes the substrate and produces a colored product. The degranulation activity of the mast cells was measured as the percentage release of enzymes outside the cell normalized with the total enzyme content in the cell.

研究目标化学品效果的肥大细胞脱颗粒测定

Source: Tsvilovskyy, V., et al. Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Assays and Functional Profiling

A Technique to Isolate Primary Mast Cells from a Murine Peritoneal Cavity

Source: Tsvilovskyy, V., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57222">Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays.</a> J. Vis. Exp. (2018).
This video demonstrates a peritoneal lavage method for isolating mature connective tissue-type mast cells from the peritoneal cavity of adult mice. Upon isolating a mixture of primary immune cells from the peritoneal cavity, selective proliferation of mast cells is performed under appropriate culture conditions.

腹膜源性肥大细胞的分离及其功能特征的 Ca^{2 +}成像和脱粒测定

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