Name: in vivo photolabeling of cells in the colon to assess migratory potential of hematopoietic cells in neonatal mice
Uploaded: 2018-08-10T14:45:00.000+00:00
Duration: 8 min 39 s
Description: 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

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>

<ol>
	<li>Maynard, C. L., Elson, C. O., Hatton, R. D., Weaver, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+interactions+of+the+intestinal+microbiota+and+immune+system.">Reciprocal interactions of the intestinal microbiota and immune system.</a> Nature. 489, 231-241 (2012).</li><li>Paun, A., Yau, C., Danska, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Influence+of+the+Microbiome+on+Type+1+Diabetes.">The Influence of the Microbiome on Type 1 Diabetes.</a> Journal of Immunology. 198, 590-595 (2017).</li><li>Mathis, D., Benoist, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiota+and+autoimmune+disease:+the+hosted+self.">Microbiota and autoimmune disease: the hosted self.</a> Cell Host Microbe. 10, 297-301 (2011).</li><li>Kollmann, T. R., Kampmann, B., Mazmanian, S. K., Marchant, A., Levy, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+the+Newborn+and+Young+Infant+from+Infectious+Diseases:+Lessons+from+Immune+Ontogeny.">Protecting the Newborn and Young Infant from Infectious Diseases: Lessons from Immune Ontogeny.</a> Immunity. 46, 350-363 (2017).</li><li>Jain, N., Walker, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet+and+host-microbial+crosstalk+in+postnatal+intestinal+immune+homeostasis.">Diet and host-microbial crosstalk in postnatal intestinal immune homeostasis.</a> Nature Reviews in Gastroenterology and Hepatology. 12, 14-25 (2015).</li><li>Mowat, A. M., Agace, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+specialization+within+the+intestinal+immune+system.">Regional specialization within the intestinal immune system.</a> Nature Reviews in Immunology. 14, 667-685 (2014).</li><li>Macpherson, A. J., Uhr, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+protective+IgA+by+intestinal+dendritic+cells+carrying+commensal+bacteria.">Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria.</a> Science. 303, 1662-1665 (2004).</li><li>Mowat, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+basis+of+tolerance+and+immunity+to+intestinal+antigens.">Anatomical basis of tolerance and immunity to intestinal antigens.</a> Nature Reviews in Immunology. 3, 331-341 (2003).</li><li>Diehl, G. E., 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=Microbiota+restricts+trafficking+of+bacteria+to+mesenteric+lymph+nodes+by+CX(3)CR1(hi)+cells.">Microbiota restricts trafficking of bacteria to mesenteric lymph nodes by CX(3)CR1(hi) cells.</a> Nature. 494 (3), 116-120 (2013).</li><li>Pham, A. H., McCaffery, J. M., Chan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+lines+with+photo-activatable+mitochondria+to+study+mitochondrial+dynamics.">Mouse lines with photo-activatable mitochondria to study mitochondrial dynamics.</a> Genesis. 50, 833-843 (2012).</li><li>Conway, K. L., 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=ATG5+regulates+plasma+cell+differentiation.">ATG5 regulates plasma cell differentiation.</a> Autophagy. 9, 528-537 (2013).</li><li>Buzoni-Gatel, D., Lepage, A. C., Dimier-Poisson, I. H., Bout, D. T., Kasper, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+transfer+of+gut+intraepithelial+lymphocytes+protects+against+murine+infection+with+Toxoplasma+gondii.">Adoptive transfer of gut intraepithelial lymphocytes protects against murine infection with Toxoplasma gondii.</a> Journal of Immunology. 158, 5883-5889 (1997).</li><li>Guo, X., Muite, K., Wroblewska, J., Fu, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+Adoptive+Transfer+of+Group+3+Gut+Innate+Lymphoid+Cells.">Purification and Adoptive Transfer of Group 3 Gut Innate Lymphoid Cells.</a> Methods in Molecular Biology. 1422, 189-196 (2016).</li><li>Morton, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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><tbody><tr><td>&lt;strong&gt;Laser&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Light Emitting Diode (LED)</td><td>THORLABS</td><td>M405FP1</td><td>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><td>LED driver</td><td>THORLABS</td><td>LEDD1B</td><td>Drives a constant current of 1200 mA through the laser. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2616</td></tr><tr><td>Optogenetics patch cable</td><td>THORLABS</td><td>M87L01</td><td>1 m long cable with an SMA connector. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=11405&amp;amp;pn=M87L01#11454</td></tr><tr><td>Fiber optic cannula</td><td>Doric lenses&amp;nbsp;</td><td>MFC_480/500-0.5_5mm_ZF1.25_C45</td><td>5 mm long cannula with an outer diameter of 500 &amp;micro;m and an inner diameter of 480 &amp;micro;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><td>Power supply</td><td>THORLABS</td><td>KPS101</td><td>Supplies 15 V with a current of 2.4 A https://www.thorlabs.com/search/thorsearch.cfm?search=KPS101</td></tr><tr><td>LG3 laser safety goggles&amp;nbsp;</td><td>THORLABS</td><td>LG3</td><td>Orange lenses with 47% visible light transmission https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=762&amp;amp;pn=LG3#2523</td></tr><tr><td>Red light&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>74327-10</td><td>15 W lamp https://us.vwr.com/store/product/12360027/paterson-safelight-electron-microscopy-sciences</td></tr><tr><td>&lt;strong&gt;Intestinal cell isolation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Patterson Veterinary</td><td>07-893-1389</td><td>CAUTION: inhalation of this anesthetic may cause dizziness, drowsiness, or even unconsciousness. This anesthetic should be used in a Class II hood.&amp;nbsp; https://www.pattersonvet.com/Supplies/ProductFamilyDetails/PIF_762328?carouselPageNumber=3</td></tr><tr><td>1X HBSS</td><td>Gibco</td><td>14025076</td><td>Ca/Mg free https://www.fishersci.com/shop/products/gibco-hbss-calcium-magnesium-no-phenol-red-4/14025076?searchHijack=true&amp;amp;searchTerm=14025076&amp;amp;searchType=RAPID&amp;amp;matchedCatNo=14025076</td></tr><tr><td>Calf Serum</td><td>Hyclone AZM</td><td>197696</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen</td><td>15575020</td><td>0.5 M concentration https://www.thermofisher.com/order/catalog/product/15575020?SID=srch-srp-15575020</td></tr><tr><td>DTT</td><td>Sigma</td><td>10197777001</td><td>CAUTION: harmful if swallowed and causes skin irritation. 1 M concentration https://www.sigmaaldrich.com/catalog/product/roche/dttro?lang=en&amp;amp;region=US</td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630080</td><td>1 M concentration https://www.thermofisher.com/order/catalog/product/15630080?SID=srch-hj-15630080</td></tr><tr><td>Petri dish</td><td>Corning</td><td>353004</td><td>https://www.fishersci.com/shop/products/falcon-easy-grip-tissue-culture-dishes-2/08772f?searchHijack=true&amp;amp;searchTerm=08772F&amp;amp;searchType=RAPID&amp;amp;matchedCatNo=08772F</td></tr><tr><td>70 micron cell strainer</td><td>Falcon</td><td>352350</td><td>https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087712</td></tr><tr><td>Micro magnetic stir bar</td><td>Fisherbrand</td><td>1451364</td><td>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><td>Magnetic stir plate</td><td>Corning Laboratory Stirrers</td><td>440826</td><td>https://www.coleparmer.com/i/corning-440826-nine-position-stirrer-120-vac-60-hz/8430420?PubID=UX&amp;amp;persist=true&amp;amp;ip=no&amp;amp;gclid=CjwKCAiAqbvTBRAPEiwANEkyCLPLrWABXmOUI0QE53NLV0Owxlcs2V1K6rWbRPOwlcVVDq000FBiQxoCqQAQAvD_BwE</td></tr><tr><td>Collagenase</td><td>Roche</td><td>5401020001</td><td>https://www.sigmaaldrich.com/catalog/product/roche/05401020001?lang=en&amp;amp;region=US&amp;amp;gclid=CjwKCAiAjuPRBRBxEiwAeQ2QPhE44qlvxjmo1PYu3zCas3w-_d6P9gKjXW82-c1EOm6NjPHCc5WuixoC_0IQAvD_BwE</td></tr><tr><td>DNase I</td><td>Sigma</td><td>10104159001</td><td>https://www.sigmaaldrich.com/catalog/product/roche/10104159001?lang=en&amp;amp;region=US</td></tr><tr><td>1X PBS</td><td>Gibco</td><td>20012-027</td><td>https://www.thermofisher.com/order/catalog/product/20012027?SID=srch-hj-20012-027</td></tr><tr><td>Pipet aid</td><td>Thermo Scientific</td><td>14387165</td><td>https://www.fishersci.com/shop/products/s1-pipette-fillers/14387165#?keyword=14387165</td></tr><tr><td>10 mL serological pipet</td><td>Falcon</td><td>357530</td><td>https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659</td></tr><tr><td>25 mL serological pipet</td><td>Falcon</td><td>357515</td><td>https://www.fishersci.com/shop/products/falcon-serological-pipets-bulk-pack-5/p-163659</td></tr><tr><td>15 mL conical centrifuge tube</td><td>Thermo Scientific</td><td>339651</td><td>https://www.thermofisher.com/order/catalog/product/339650</td></tr><tr><td>50 mL conical centrifuge tube</td><td>Thermo Scientific</td><td>339653</td><td>https://www.thermofisher.com/order/catalog/product/339650</td></tr><tr><td>&lt;strong&gt;Single cell suspension&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Eppendorf tubes</td><td>Seal-Rite</td><td>1615-5500</td><td>Holds 1.5 mL. https://www.usascientific.com/Seal-Rite-1.5-ml-tube.aspx</td></tr><tr><td>Tissue homogenizer&amp;nbsp;</td><td>Kimble</td><td>K7495400000</td><td>Requires 2 AA batteries. https://www.fishersci.com/shop/products/kontes-pellet-pestle-cordless-motor-cordless-motor/k7495400000</td></tr><tr><td>Homogenizer tips&amp;nbsp;</td><td>Kimble</td><td>7495210590</td><td>Plastic, 0.5 mL tips https://www.fishersci.com/shop/products/kimble-chase-kontes-pellet-pestle-14/k7495210590#?keyword=7495210590</td></tr><tr><td>ACK lysing buffer</td><td>Gibco</td><td>A10492-01</td><td>https://www.thermofisher.com/order/catalog/product/A1049201?SID=srch-hj-A10492-01</td></tr><tr><td>40 micron cell strainer</td><td>Falcon</td><td>08-771-1</td><td>https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087711</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BV786 anti-mouse CD45&amp;nbsp;</td><td>BD</td><td>564225</td><td>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><td>Live/Dead</td><td>Invitrogen</td><td>L34962</td><td>https://www.thermofisher.com/order/catalog/product/L34962</td></tr><tr><td>&lt;strong&gt;Other&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Razor blades</td><td>VWR</td><td>55411-050</td><td>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 ...

in-vivo-photolabeling-cells-colon-to-assess-migratory-potential

Research

JoVE Journal

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 Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

科研

发育生物学

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

生物学

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

使用<em&gt;在体内</em&gt;影像监测进展的实验小鼠巨细胞病毒感染的新生儿

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

免疫与感染

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

功能研究和过继转移实验小鼠骨髓嗜中性粒细胞的分离，纯化及贴标

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

一种用于乳腺肿瘤微环境活体内成像的无标记分割方法

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

癌症研究

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

跟踪小鼠骨髓单核细胞<em&gt;在体内</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

用于研究小鼠视网膜循环中流动动力学的红细胞和白细胞的荧光染料标记

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

医学

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...