尼亚耆那得到了 NIH/NIAID 职业转型奖1K22AI116661-01 的支持。

所有动物程序都是在马萨诸塞州总医院的机构动物护理和使用委员会 (IACUC) 批准和遵守的情况下进行的。
注意: 此协议涉及使用3b 类激光 (LG3)。LG3 激光安全护目镜在操作激光时必须始终使用。必须遵循适当的培训和安全准则, 以避免受伤的危险。
1. 激光的设计与装配
<ol><li>通过连接到 led 的4针 M8 连接器电缆, 将发光二极管 (LED) (405 nm、19.3 兆瓦、1400 mA、光纤耦合) 连接到 led 驱动程序。小心拧紧螺钉以固定连接。</li>
	<li>在将光遗传学补丁电缆连接到 led 之前, 请按住指示灯的连接器打开至指示灯, 以确保没有可见障碍物。吹孔, 以消除任何障碍, 如果必要的, 并插入光遗传学补丁电缆进入开口。</li>
	<li>通过将套管插入连接器套筒的任一端, 将光纤套管连接到光遗传学贴片电缆。将连接器套筒的另一端连接到修补电缆。 施加足够的压力, 以确保连接, 但要小心不要弯曲或打破在套管中的光纤。 注: 当激光不使用时, 脆弱的光纤套管被保护罩覆盖。</li>
	<li>将 LED 驱动程序设置为连续波 (CW) 模式, 连续调整旋钮为 0, 当前限制设置为 1.2 A。</li>
</ol>2. 结肠中细胞的 Photoconversion
注: 雄性和雌性小鼠在出生后暴露于 intracolonic 405 nm, 1-2 天, 在1岁之前被献祭。
<ol><li>安全的一个黑暗的位置, 进入一个电源插座。将 LED 驱动程序插入插座。 注意: 这一过程是在最小的白光, 因为长时间暴露的小鼠对周围的光可能导致不必要的 photoconversion 细胞。红灯被用作替代品。 注意: 以下步骤涉及使用动物麻醉剂。麻醉的管理发生在二类敞篷或使用另一个适当的清除系统。吸入麻醉药可能会引起头晕、嗜睡甚至昏迷。</li>
	<li>麻醉 4-5% 异氟醚在感应盒 100% O2 中接触到的小鼠。通过紧捏后爪 (无反应) 来验证适当的麻醉深度。通过鼻锥在大约2% 异氟醚保持麻醉。使用适当的罩或麻醉剂清除装置, 防止处理程序暴露于异氟醚。</li>
	<li>使用1只手用食指和拇指将鼠标的背部轻轻地背在 intracolonic 上, 将麻醉鼠标放置在其上。把鼠标打开, 露出腹部。松开手柄, 如果鼠标开始失去其粉红色的颜色或它的呼吸速度开始减慢。在圆环和小手指之间保护鼠标的尾部。</li>
	<li>另一方面, 从套管上取下防护帽。在保持套管与鼠标中线平行的长轴时, 轻轻地将光纤套管插入肛门内, 以便所有5毫米光纤都在鼠标内。 注意: 本程序执行缓慢, 小心不要穿孔结肠壁。分钟摆动和扭曲的套管帮助推进到结肠。</li>
	<li>戴上安全护目镜, 将激光电流增加到最大值 (顺时针方向)。通过观察鼠标的半透明腹部来确认紫外线的存在。 注: LG3 激光安全护目镜显著降低能见度。有一个同事 (佩戴防护设备) 协助戴上护目镜并打开激光。</li>
	<li>将冒号暴露在激光照射下, 共2分钟. 每三十年代取出套管大约1毫米, 以最大限度地扩大暴露在光线下的腔区。在2分钟结束时, 关掉激光。</li>
	<li>慢慢地从鼠标中取出套管。在一个空温暖的笼子里从麻醉中恢复后, 把老鼠送回家庭笼子。</li>
</ol>3. 肠道淋巴细胞的分离
<ol><li>准备 1 L 的汉克平衡盐溶液/小牛血清 (HBSS/CS) 缓冲, 200 毫升的上皮带缓冲, 500 毫升的洗涤介质, 200 毫升的乙二胺四乙酸 (EDTA) 无洗涤介质。 注: 试剂卷的计算样本大小为9。<ol>
<li>通过增加50毫升的 cs 到950毫升的 HBSS, 使 HBSS/cs 缓冲器 (HBSS; 5% cs)。把它储存在冰上。</li>
		<li>准备上皮带缓冲液 [HBSS; 5% CS; 1 毫米 dithiothreitol (德勤); 5 毫米 edta; 15 毫米 4-(2-羟乙基) piperazine-1-ethanesulfonic 酸 (HEPES) (pH 7.2-7.5)] 通过加入10毫升的 CS, 200 µL 的 1 m 的, 毫升的 EDTA, 2 毫升的0.5 米的 HEPES 缓冲到3毫升的 HBSS。将上皮带缓冲剂放置在37摄氏度的水浴中。</li>
		<li>通过增加5毫升的 cs 和1毫升的0.5 米 edta 到494毫升的 HBSS, 使洗涤介质 (HBSS; 1% cs; 1 毫米 edta)。把它储存在室温下。</li>
		<li>通过加入2毫升的 cs 和3毫升的1米 HEPES 至195毫升 HBSS, 制备无 EDTA 洗涤介质 (HBSS; 1% cs; 15 毫米 HEPES)。把它储存在室温下。</li>
		<li>制作一个外地资产管制署缓冲区 [1x 磷酸盐缓冲盐水 (PBS) (pH = 7.2); 2% cs] 通过增加10毫升 cs 到490毫升 PBS。把它储存在冰上。</li>
	</ol></li>
	<li>在 60 x 10 毫米培养皿内放置70µm 细胞过滤器, 准备组织收集皿。添加10毫升的 HBSS/CS 缓冲器 (HBSS; 5% cs 缓冲器在步骤3.1.1 和储存在冰上) 到盘子里, 放在冰上。</li>
	<li>弄死鼠标通过过量吸入异氟醚使用适当的麻醉清除 (约3分钟接触 > 4% 氟醚在室内空气或 100% O2)。在明显停止呼吸和反射运动后, 通过牢牢捏一只后爪 (建议使用镊子) 来验证是否没有深部疼痛。使用一双手术剪刀或一把新的直剃刀, 确保安乐死通过斩首。 注: 由于动物的大小和年龄, 不建议在这一步骤中颈椎脱位。这些方法符合 2013 AVMA 关于安乐死的指导原则, 并得到 MGH IACUC 的批准。</li>
	<li>用干净的钳子和剪刀收割结肠, 打开腹部。将肠道反射到老鼠腹部的1侧露出结肠。首先 transecting 它的远端 (向肛门) 切除整个结肠到盲肠。然后, 用钳子牢牢抓住结肠, 用剪刀切开尿道到直肠。把结肠尽可能的靠近肛门。取出结肠后取脾 (见下面步骤 4.1-4.2)。 注意: 虽然 photoconversion 协议只针对新生儿结肠的 25% (5 毫米的光纤套管), 但整个结肠的收获是为了帮助细胞隔离。</li>
	<li>用镊子将结肠中的内容从结肠中推到干纸巾上, 以清除冒号。融汇用剃刀刀片将结肠细砍成一个培养基的盖子上的糊状, 然后将这个浆糊转移到培养皿中的细胞过滤器上。</li>
	<li>将清洁的 8 mm 长的磁力搅拌棒添加到过滤器上, 将盘子放在9位磁性搅拌器板上。将磁搅拌棒速度设置为 800 rpm, 室温下孵化细胞5分钟。丢弃缓冲区并重复使用10毫升的 HBSS/CS (HBSS; 5% 级 cs 缓冲器, 在步骤3.1.1 和储存在冰上)。在洗涤结束时丢弃缓冲区。</li>
	<li>添加10毫升的 prewarmed 上皮带缓冲液 (HBSS) 游离上皮细胞; 5% CS; 1mM. 5mM EDTA; 15mM HEPES [pH 7.2-7.5] 缓冲器, 是在步骤3.1.2 和存储在37°c 水浴) 到细胞过滤器和搅拌 800 rpm 30 分钟在37°c 孵化器。在孵化结束时, 将上皮细胞中丰富的缓冲液转移到干净的15毫升管中。 注意: 上皮细胞和相关细胞 (肠道上皮干细胞和肠道上皮淋巴细胞) 被释放到缓冲区中, 而在本步骤11中, 完整的固有层中的细胞仍保留在细胞过滤器中。<ol>
<li>在室温下离心浓缩肠上皮分数 (IELs) 在 700 x g 5 分钟。丢弃上清液, 并在1毫升的并用重悬中对细胞颗粒进行去除。储存在冰上, 以便在步骤3.15 中使用。</li>
	</ol></li>
	<li>在细胞过滤器2x 内, 用 HBSS/CS 洗净叶片固有部分。为此, 添加10毫升的 HBSS/CS 解决方案 (HBSS; 5% cs 缓冲区, 这是在步骤 3.1.1, 已储存在冰上) 到细胞过滤器, 并搅拌在 800 rpm 5 分钟在37°c 孵化器。每次清洗后丢弃缓冲区。</li>
	<li>在洗涤介质中清洗样品2x。添加10毫升的洗涤介质 (HBSS; 1% CS; 1 毫米 EDTA 缓冲器, 在步骤3.1.3 和储存在室温下) 到细胞过滤器, 并在15摄氏度孵化器中以 800 rpm 为37分钟搅拌。每次清洗后丢弃缓冲区。<ol>
<li>使用洗涤介质后, 将其存储在冰上以供步骤3.14 使用。</li>
	</ol></li>
	<li>用 HBSS/cs 清洗样品 2x. 添加10毫升的 HBSS/cs 缓冲器 (HBSS; 5% cs 缓冲器, 在步骤3.1.1 已储存在冰上) 到细胞过滤器, 并在37°c 孵化器 800 rpm, 以5分钟。每次清洗后丢弃缓冲区。</li>
	<li>在无 EDTA 洗涤缓冲液中孵化组织。加入10毫升无 EDTA 洗涤缓冲液 (HBSS; 1% CS; 15 毫米 HEPES 缓冲器, 在3.1.4 和室温下储存) 到细胞过滤器中, 在室温下将板材孵化为10分钟而不搅拌。在该步骤的末尾丢弃缓冲区。</li>
	<li>用 HBSS/CS 冲洗样品 2x, 并准备酶溶液。添加10毫升的 HBSS/CS (HBSS; 5% cs 缓冲器, 在步骤 3.1.1, 已储存在冰上) 缓冲区到细胞过滤器, 并在37°c 孵化器 800 rpm, 以10分钟的搅拌。每次清洗后丢弃缓冲区。<ol>
<li>制备酶溶液 (无 edta 洗涤缓冲器; 0.1 毫克/毫升 DNase I; 167 毫克/毫升胶原酶) 在第二洗涤期间: 添加9毫克的 DNase I 和15毫克的冻干胶原酶, 以90毫升无 EDTA 洗涤缓冲。在室温下存储解决方案。</li>
	</ol></li>
	<li>消化细胞过滤器内的固有层含量, 获得固有的细胞膜 (LPLs)。添加10毫升的酶溶液 (无 EDTA 洗涤缓冲器; 0.1 毫克/毫升 DNase I; 167 毫克/毫升胶原酶缓冲液在步骤3.12.1 和储存在室温下) 到细胞过滤器, 并搅拌在 800 rpm 45 分钟在37°c 孵化器。 注: LPLs 在酶消化后释放到溶液中。<ol>
<li>收集含有细胞的酶解, 并将其通过新的细胞过滤器滤入50毫升锥形管中。</li>
	</ol></li>
	<li>加入20毫升的冷洗介质 (HBSS; 1% CS; 1mM EDTA 缓冲液, 3.1.3 步进3.9.1 中的冰上, 储存在台阶上的冰块中), 以阻断酶解中的胶原酶活性。在室温下离心管在 700 x g 处6分钟。在1毫升的并用重悬中, 将含有 LPLs 的颗粒丢弃上清液。</li>
	<li>结合 IEL (从步骤 3.7.1) 和 LPL (从步骤 3.14) 分数和染色他们与荧光共轭抗体为流细胞分析 (见步骤 5)。 注: 如果需要间隔信息, IEL 和 LPL 分数可能会被单独染色。</li>
</ol>4. 从脾脏分离淋巴细胞
<ol><li>在离心管中加入500µL 的内流器缓冲器, 准备收集管。把它储存在冰上。</li>
	<li>除去结肠 (步骤 3.4) 后, 收获整个脾脏并将其放在收集管中。把纸巾储存在冰上。</li>
	<li>使用电组织均质器, 融汇脾在离心管约1分钟离心机在 300 x g [微离心机] 在室温下5分钟, 并吸取上清。</li>
	<li>通过 resuspending 500 µL 铵-氯-钾 (ACK) 裂解缓冲液中的样品进行红细胞裂解, 以渗透裂解启动细胞死亡。允许样品在室温下孵化 1-2 分钟, 然后将其并用重悬725µL。</li>
	<li>离心样品在 300 x g [微离心机] 在室温下5分钟, 并吸取上清。并用重悬500µL 中的颗粒, 并将其通过40µm 细胞过滤器过滤成一个新的离心管。将整个样品转移到一个新的离心管, 并用荧光共轭抗体对其进行染色细胞分析 (见步骤 5)。</li>
</ol>5. 用流式细胞仪识别 Dendra 细胞
<ol><li>离心的 IEL/LPL 和脾脏样品在 300 x g [微离心机] 5 分钟室温和吸出上清。</li>
	<li>并用重悬100µL 抗体染色混合体中的颗粒。通过将荧光共轭 anti-CD45 抗体的1µL 添加到每个样品的100µL, 以制备抗体染色组合。 注意: 在实验之前滴定流式细胞术中的抗体是很重要的, 以确定结合饱和度发生的最佳浓度。</li>
	<li>在冰上孵化样品35分钟 (在黑暗的地方)。</li>
	<li>并用重悬300µL 的样品, 并将其离心机在 300 x g [微离心机] 中, 室温下5分钟。在325µL 的并用重悬中吸出上清液和样品。分析流式细胞仪上的样品。</li>
</ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+photoconversion+reveals+unexpectedly+broad+leukocyte+trafficking+to+and+from+the+gut.">Endoscopic photoconversion reveals unexpectedly broad leukocyte trafficking to and from the gut.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 6696-6701 (2014).</li></ol>

作者没有什么可透露的。

细胞的识别和鉴定与结肠菌群的影响是重要的, 应该有助于理解如何将信息从粘膜微环境传递到身体的其余部分。研究肠道相关细胞迁移的一种方法, 需要将肠道相关细胞分离出来, 然后将移植到受体小鼠体内, 以确定它们的组织自导模式和功能12,13。此方法是有限的, 因为仅可以跟踪传输的特定单元格类型。此外, 由于从组织中分离细胞和纯化过程而产生...

哺乳动物胃肠道有数以百计的细菌存在于与宿主1的共生关系中。在当地环境中存在的免疫细胞加强了与这些微生物的和平共处, 并对病菌入侵建立了保护性屏障。因此, 免疫细胞和微生物群之间的双向相互作用是建立一个共生社区, 教育宿主免疫系统并设定免疫反应性的阈值的关键。微生物组成的变化, 或 dysbiosis, 可能扰乱免疫动态平衡和扰乱调节电路, 抑制肠道炎症导致免疫介导的疾病, 如1型糖尿病和 IBD2,3.
出生后的时间是一个独特的发展窗口, 肠道微生物群落开始建立的同时免疫系统成熟4。产后微生物群是不稳定的, 与转移在社区构成自然地和频繁地发生5。与微生物群相互作用的免疫细胞位于肠道的两个不同的解剖位置--固有层和肠道上皮6。许多类型的免疫细胞存在于肠道, 包括淋巴细胞 (如 T 细胞, B 细胞, 和先天淋巴细胞) 以及髓细胞 (包括树突状细胞, 单核, 和巨细胞)。这些细胞, 也称为造血细胞, 执行多种功能, 保持肠道屏障和维持动态平衡。
除了在肠道部位的调节功能外, 粘膜的免疫细胞还可能携带微生物信息到肠外的部位, 以调节全身免疫7、8、9。这是一个日益增长的研究兴趣的领域, 并强调需要的方法, 以确定免疫细胞迁移出肠道组织, 以探讨其功能。这里报告的协议使用了一个商业上可用的老鼠模型, 其中 photoconvertible 荧光蛋白被利用来标记细胞。范切除小鼠无所不在表达绿色荧光 Dendra2 蛋白, 是不可逆转地切换到红色荧光激活时紫外线 (UV) 光10。利用光纤套管将405纳米光传送到新生小鼠结肠中, 我们证明了 photoconverted 造血细胞 (起源于或通过结肠过渡) 可以在脾脏中找到。

<table border="0" cellpadding="0" cellspacing="0" style="width:873px;" width="874">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Laser</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
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			<td height="168" style="height:168px;width:259px;">Light Emitting Diode (LED)</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">M405FP1</td>
			<td style="width:247px;">CAUTION: this is a Class 3b laser. Safety goggles must be worn when using the laser. It emits a 405 nm wavelength with a current of 1400 mA. It is fiber-coupled. It accepts SMA connector. https://www.thorlabs.com/thorproduct.cfm?partnumber=M405FP1</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">LED driver</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">LEDD1B</td>
			<td style="width:247px;">Drives a constant current of 1200 mA through the laser. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2616</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:259px;">Optogenetics patch cable</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">M87L01</td>
			<td style="width:247px;">1 m long cable with an SMA connector. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=11405&#38;pn=M87L01#11454</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">Fiber optic cannula</td>
			<td style="width:201px;">Doric lenses&#160;</td>
			<td style="width:167px;">MFC_480/500-0.5_5mm_ZF1.25_C45</td>
			<td style="width:247px;">5 mm long cannula with an outer diameter of 500 &#181;m and an inner diameter of 480 &#181;m. The NA value is 0.5. The ferrule is zirconia, 1.25 mm OD. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=6036</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">Power supply</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">KPS101</td>
			<td style="width:247px;">Supplies 15 V with a current of 2.4 A https://www.thorlabs.com/search/thorsearch.cfm?search=KPS101</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:259px;">LG3 laser safety goggles&#160;</td>
			<td style="width:201px;">THORLABS</td>
			<td style="width:167px;">LG3</td>
			<td style="width:247px;">Orange lenses with 47% visible light transmission https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=762&#38;pn=LG3#2523</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Red light&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>74327-10</td>
			<td style="width:247px;">15 W lamp https://us.vwr.com/store/product/12360027/paterson-safelight-electron-microscopy-sciences</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Intestinal cell isolation</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Isoflurane</td>
			<td style="width:201px;">Patterson Veterinary</td>
			<td style="width:167px;">07-893-1389</td>
			<td style="width:247px;">CAUTION: inhalation of this anesthetic may cause dizziness, drowsiness, or even unconsciousness. This anesthetic should be used in a Class II hood.&#160; https://www.pattersonvet.com/Supplies/ProductFamilyDetails/PIF_762328?carouselPageNumber=3</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">1X HBSS</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">14025076</td>
			<td style="width:247px;">Ca/Mg free https://www.fishersci.com/shop/products/gibco-hbss-calcium-magnesium-no-phenol-red-4/14025076?searchHijack=true&#38;searchTerm=14025076&#38;searchType=RAPID&#38;matchedCatNo=14025076</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Calf Serum</td>
			<td style="width:201px;">Hyclone AZM</td>
			<td style="width:167px;">197696</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">EDTA</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:167px;">15575020</td>
			<td style="width:247px;">0.5 M concentration https://www.thermofisher.com/order/catalog/product/15575020?SID=srch-srp-15575020</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:259px;">DTT</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:167px;">10197777001</td>
			<td style="width:247px;">CAUTION: harmful if swallowed and causes skin irritation. 1 M concentration https://www.sigmaaldrich.com/catalog/product/roche/dttro?lang=en&#38;region=US</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">HEPES</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">15630080</td>
			<td style="width:247px;">1 M concentration https://www.thermofisher.com/order/catalog/product/15630080?SID=srch-hj-15630080</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:259px;">Petri dish</td>
			<td style="width:201px;">Corning</td>
			<td style="width:167px;">353004</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-easy-grip-tissue-culture-dishes-2/08772f?searchHijack=true&#38;searchTerm=08772F&#38;searchType=RAPID&#38;matchedCatNo=08772F</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">70 micron cell strainer</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">352350</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087712</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Micro magnetic stir bar</td>
			<td style="width:201px;">Fisherbrand</td>
			<td style="width:167px;">1451364</td>
			<td style="width:247px;">Rinse in 70% ethanol after each use. Rinse several times in distilled water prior to each use. The bar is 8 mm long with an octagonal shape. https://www.fishersci.com/shop/products/fisherbrand-octagonal-magnetic-stir-bars-12/1451364#?keyword=1451364</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:259px;">Magnetic stir plate</td>
			<td style="width:201px;">Corning Laboratory Stirrers</td>
			<td style="width:167px;">440826</td>
			<td style="width:247px;">https://www.coleparmer.com/i/corning-440826-nine-position-stirrer-120-vac-60-hz/8430420?PubID=UX&#38;persist=true&#38;ip=no&#38;gclid=CjwKCAiAqbvTBRAPEiwANEkyCLPLrWABXmOUI0QE53NLV0Owxlcs2V1K6rWbRPOwlcVVDq000FBiQxoCqQAQAvD_BwE</td>
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		<tr height="147">
			<td height="147" style="height:147px;width:259px;">Collagenase</td>
			<td style="width:201px;">Roche</td>
			<td style="width:167px;">5401020001</td>
			<td style="width:247px;">https://www.sigmaaldrich.com/catalog/product/roche/05401020001?lang=en&#38;region=US&#38;gclid=CjwKCAiAjuPRBRBxEiwAeQ2QPhE44qlvxjmo1PYu3zCas3w-_d6P9gKjXW82-c1EOm6NjPHCc5WuixoC_0IQAvD_BwE</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">DNase I</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:167px;">10104159001</td>
			<td style="width:247px;">https://www.sigmaaldrich.com/catalog/product/roche/10104159001?lang=en&#38;region=US</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">1X PBS</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">20012-027</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/20012027?SID=srch-hj-20012-027</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:259px;">Pipet aid</td>
			<td style="width:201px;">Thermo Scientific</td>
			<td style="width:167px;">14387165</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/s1-pipette-fillers/14387165#?keyword=14387165</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">10 mL serological pipet</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">357530</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">25 mL serological pipet</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">357515</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">15 mL conical centrifuge tube</td>
			<td style="width:201px;">Thermo Scientific</td>
			<td style="width:167px;">339651</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/339650</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">50 mL conical centrifuge tube</td>
			<td style="width:201px;">Thermo Scientific</td>
			<td style="width:167px;">339653</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/339650</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Single cell suspension</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">Eppendorf tubes</td>
			<td style="width:201px;">Seal-Rite</td>
			<td style="width:167px;">1615-5500</td>
			<td style="width:247px;">Holds 1.5 mL. https://www.usascientific.com/Seal-Rite-1.5-ml-tube.aspx</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:259px;">Tissue homogenizer&#160;</td>
			<td style="width:201px;">Kimble</td>
			<td>K7495400000</td>
			<td style="width:247px;">Requires 2 AA batteries. https://www.fishersci.com/shop/products/kontes-pellet-pestle-cordless-motor-cordless-motor/k7495400000</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:259px;">Homogenizer tips&#160;</td>
			<td style="width:201px;">Kimble</td>
			<td style="width:167px;">7495210590</td>
			<td style="width:247px;">Plastic, 0.5 mL tips https://www.fishersci.com/shop/products/kimble-chase-kontes-pellet-pestle-14/k7495210590#?keyword=7495210590</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">ACK lysing buffer</td>
			<td style="width:201px;">Gibco</td>
			<td style="width:167px;">A10492-01</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/A1049201?SID=srch-hj-A10492-01</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">40 micron cell strainer</td>
			<td style="width:201px;">Falcon</td>
			<td style="width:167px;">08-771-1</td>
			<td style="width:247px;">https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087711</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:873px;">Antibodies</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:259px;">BV786 anti-mouse CD45&#160;</td>
			<td style="width:201px;">BD</td>
			<td style="width:167px;">564225</td>
			<td style="width:247px;">Clone 3O-F11 https://www.bdbiosciences.com/us/reagents/research/antibodies-buffers/immunology-reagents/anti-mouse-antibodies/cell-surface-antigens/bv786-rat-anti-mouse-cd45-30-f11/p/564225</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Live/Dead</td>
			<td>Invitrogen</td>
			<td>L34962</td>
			<td style="width:247px;">https://www.thermofisher.com/order/catalog/product/L34962</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Other</td>
			<td style="width:201px;"></td>
			<td style="width:167px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Razor blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td style="width:247px;">Use for decapitation. https://us.vwr.com/store/product/4548306/vwr-razor-blades</td>
		</tr>
	</tbody>
</table>

in vivo photolabeling of cells in the colon to assess migratory potential of hematopoietic cells in neonatal mice

肠道细菌群落是早期建立的, 对免疫细胞的发育和功能有影响。新生儿微生物群易受多种外部影响, 包括抗生素的使用和饮食, 这会影响对自身免疫和炎症性疾病的敏感性。炎症性肠病 (IBD) 等疾病的特点是大量的免疫细胞涌入肠道。然而, 由微生物群所调节的免疫细胞可能会另外从肠道中移出, 以影响肠道的免疫应答。因此, 有必要识别和鉴定可能携带微生物信息从肠道到远端部位的细胞。在这里, 我们描述了一种方法来标记细胞在结肠的新生小鼠在体内, 使他们的身份, 在肠外的地方, 迁移后鉴定。

用光纤电缆将405纳米光传送到2天的老范切除小鼠的冒号中。在以前的实验中, 三十年代的暴露被确定为最大 photoconversion 的结肠细胞具有最小的细胞毒性 (图 1a)。因此, 按照《议定书》的规定, 对结肠的不同部分进行连续三十年代的曝光。激光照射后, 老鼠立即被安乐死, 细胞的 photoconversion 被确定。采用荧光共轭 anti-CD45 抗体对全?...

Watch this Scientific Journal Video about In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice at JoVE.com

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

这里描述的协议使用了 photolabeling 方法在新生老鼠特别辨认免疫细胞移居从结肠到肠外的站点。这一策略将有助于研究早期生命中宿主-微生物的相互作用。

体内结肠细胞 Photolabeling 对新生小鼠造血细胞迁移潜能的评价

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

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Medicine

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

5.7K 次观看.  Massachusetts General Hospital. 这里描述的协议使用了 photolabeling 方法在新生老鼠特别辨认免疫细胞移居从结肠到肠外的站点。这一策略将有助于研究早期生命中宿主-微生物的相互作用。

视频: In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

一项战略，以确定在常见的疾病，如自闭症和精神分裂症的新突变

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

科研

医学

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

C57BL/6J小鼠乳腺间质细胞和癌细胞的移植

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

在锝-99m在马腱末端标记的骨髓间充质干细胞的体内成像和跟踪

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

同基因小鼠黑色素瘤细胞注射，以确定其在肺转移潜能

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...