The authors thank M. Gordon of the Queen's University Biomedical Imaging Centre for assistance. This work was supported by operating grants from the Cancer Research Society of Canada (19439) and the Canadian Institutes for Health Research (MOP-142303) (LMM), and by Ontario Graduate Scholarships and studentships from the Terry Fox Research Institute Training Program in Transdisciplinary Cancer Research (SMM, EYL), and by a Craig Jury Summer Studentship (SMM).

注:所有试剂和耗材均列在材料清单 。 1.球体由生产细胞聚集<ol><li> 甲基纤维素溶液:准备100毫升100毫克/毫升甲基纤维素。 <ol><li>热火50毫升超纯H 2 O操作80℃。加入10克甲基纤维素粉末和搅动，直到颗粒被均匀地分散。 </li><li>带给用冷超纯的 H 2ö终体积，并在4℃下搅拌，直到溶液变得清晰，稻草色，并且不包含可见固体。 </li><li>通过0.45微米的过滤器通过除去未溶解的固体。制备的溶液可以被等分并储存于4℃多达12个月。 注:甲基纤维素用作无细胞毒性的，惰性的，悬浮剂以增强细胞 - 细胞粘附和阻止粘附细胞单层的形成。甲基纤维素是可溶于水，并且可以被洗去以下spherOID生成。 </li></ol></li><li> 球状体形成中 注意:使用前，立即准备球体形成网上平台。不要储存。 <ol><li>稀甲基纤维素溶液至1-5毫克/毫升在适当的培养基中。 </li><li>通过0.22微米的过滤器传送到消毒和去除未溶解的固体。 </li></ol></li><li> Dulbecco磷酸盐缓冲盐水（PBS） <ol><li>稀释至1x和使用之前，将通过一个0.45微米的过滤器。制备的溶液可以无限期地在室温下储存。 </li></ol></li><li> 细胞球体的生产（图1） 注:事先准备好所有试剂。由灰尘，纤维或其他不溶性微粒，以避免溶液的污染是重要的。这些污染物的存在会球体形成产生不利影响。培养基和其他解决方案应通过一个0.45微米的过滤器被传递到当POS除去这些颗粒sible。传输解决方案时，这些会带来额外的纤维避免使用棉过滤移液器。 <ol><li>培养细胞单层在适当的培养70％汇合培养基补充有10％血清。抽吸培养基并用1×PBS冲洗以除去碎片。添加3mL的胰蛋白酶-EDTA（0.05％胰蛋白酶）解离缓冲液，以10cm的细胞培养皿中，在37℃下孵育细胞和5％ 的 CO 2。 </li><li>检查显微镜下的细胞在10倍的放大倍率，以确认支队。中和分离缓冲液与7毫升血清补充培养基。 </li><li>在500×g离心10分钟，轻轻地沉淀细胞离心细胞悬浮液。 </li><li>除去上清液。使用具有过滤嘴一个P1000吸管在1mL球体形成培养基悬浮细胞沉淀。 注:细胞悬液应无团块。 <ol><li>以磨碎的细胞沉淀，压在管的底部枪头以微小的角度，而移液剪切细胞沉淀。避免捣碎超过3 - 5次，否则会损坏细胞。吸管慢慢地，也不排除枪头的全部内容，以避免产生气泡。 </li></ol></li><li>算使用血球细胞和稀释细胞悬浮液，以1×10 4个细胞/ mL。 注:细胞数可以被优化，以产生不同大小的球体。受损细胞的锥虫蓝染色可以用于确认悬浮细胞的生存力。 </li><li>转移细胞悬液至无菌的，无尘的多道移液器贮存和使用过滤嘴分配100微升到96孔U型底细胞斥板的各孔中。 <ol><li>用过滤超纯H 2Ø冲洗水库去除灰尘和纤维。 </li><li>混合周期性而接种，以防止细胞从沉降到储存器的底部的细胞悬浮液。为了避免产生气泡，使用反向移液技术，同时混合和dispensinG。 </li></ol></li><li>转移板到组织培养箱（37℃和5％的CO 2），并每日检查为球体的形成。细胞应解决的每一个的底部内6小时以及和通常聚合成球状体24-48小时（ 图2）内。 </li><li>对于成功的球体形成的确认，使用P10尖，轻轻吸取媒体在球体，而在放大10倍的显微镜下观察。适当地形成球体将从板和轧辊松开，确认其三维结构。 注:甲基纤维素和血清浓度应通过滴定来确定对于每个细胞系，以产生形成一个单一的球状体的每孔。以这种方式对选定的细胞系的优化条件示于表1，并形成在图2B的球状体的例子。球状体可生长时间超过指示但是，当他们变得更大他们的成长越来越困难处理无碎片。如果细胞形成一个单层，卫星球状体，或不沉降到井的底部，甲基纤维素和血清的浓度可能需要为最佳的球体形成（ 图3B）来进一步调整。不要使用含有污染物的球体，如纤维或灰尘（ 图3A）。预形成-球状体可以与如生长因子，活细胞的污渍，或用于分析的抑制剂的感兴趣的化合物进行治疗。 </li></ol></li></ol> 2.球体侵袭试验（图4） <ol><li> 胶原蛋白中和 <ol><li>酷所有的液体到4°C和胶原蛋白的合作以防止不必要的聚合反应时，应举行冰上。 </li><li>通过0.22微米的过滤器在超纯H 2 O.通过所有的解决方案，准备NaOH和新鲜的0.1M的新鲜的0.1 M和0.01M的解决方案。 </li><li>混合型牛胶原（3.1毫克/毫升的库存），0.01M NaOH和10倍的PBS（8:1:1的比例以体积计），并调整到使用冷0.1M NaOH中和HCl pH为7.4。胶原的最终浓度为2.5毫克/毫升。 </li><li>准备足够的中和胶原填充成像容器的2-3毫米的深度。 100微升的最小需要在材料清单中列出的8孔组织培养室滑动的每个孔中。 </li></ol></li><li> 嵌入在胶原球体（图4） <ol><li>涂层具有最小的50微升中和胶原的8孔组织培养室滑动的各孔的底部。 <ol><li>使用反向移液技术，以避免在胶原产生气泡。用枪头过孔表面均匀地涂抹胶原蛋白。 注:此胶原底层将持有悬挂的球体，通常是1-2毫米厚。基底层最小化的形成上层胶原层上的弯月面的。如果基底层太薄，球体可能使接触与腔室滑动件的底部和形成细胞单层。 </li></ol></li><li>放置玻片，并充满超纯H 2Ø35毫米的组织培养皿，在10cm的组织培养皿。在37℃下孵育直至胶原聚合，通常为1小时。请将剩余的中和冰上胶原蛋白。 注:充满水的35mm培养皿将提供湿度和防止干燥。该室幻灯片应保持在整个试验这种湿度试验箱。胶原基底层可以留聚合过夜。较长的孵育通常导致更硬的凝胶。 </li><li>使用P1,000滤嘴，收集来自96孔U型底电池斥板于1.5mL单元顶部管预形成球状体（步骤1.4.7）。吸管慢慢地，避免反复或剧烈的移液，以尽量减少球体的流体剪切。多个球状体可用于转移一个单管被汇集到8孔组织培养室滑动的一个孔中。 </li><li>离心收集球体在TABL在2000 XG 2-5秒和ETOP离心使用P200移液器上清。避免离心比需要更长的，因为这可能会导致球体掰开或聚集在一起。 注:吹打除去​​大部分上清液后，该管可以被仔细地在一个干净的纸巾吸走多余的液体反转。不要让球体干燥。 </li><li>使用大口径P200尖，轻轻悬浮在50微升中和胶原蛋白球体。任何球状体转移到室幻灯片之前做此收集球体每管。保持一直悬浮在胶原蛋白中和冰上直至准备转移到玻片的球体。 </li><li>从孵化器中取出室幻灯片，认真吸取胶原基础层顶部的球体胶原混合。在37℃和5％的CO 2，直至胶原聚合。 <ol><li>不要免除吸管的全部内容，以避免产生气泡。 Dropw伊势移液减少干扰胶原基层的机会。 </li></ol></li><li>慢慢加入至少100微升温热含有期望的趋化因子，抑制剂，或其他治疗的腔室滑动的每孔培养基中。如果液体吸到太大力球体含胶原层撕裂。培养基可缓慢分配向下一个井的侧面，以避免扰乱胶原。 </li><li>孵育室滑动，在37℃和5％的CO 2，以允许细胞侵袭。细胞侵袭到周围的胶原可通过明场或荧光成像观察和通过各种使用落射荧光，共焦或者光片显微镜方法定量。 </li></ol></li></ol> 3.入侵的量化:明场显微镜<ol><li>在设定的时间间隔，图像在10X放大率用明场显微镜（步骤2.2.8）胶原嵌入球体。 注意:长期活细胞的实验中，加热的显微镜载物台和CO 2调节的室可以用于保持在37℃ 的 CO 2而成像球状体和5％。 </li><li>使用软件如ImageJ的测量细胞侵入周围的胶原蛋白和在每个时间点入侵的平均距离的数量量化侵袭。 </li></ol> 4.定量入侵:荧光显微镜用活细胞染色<ol><li>以下步骤2.2.8，补充用荧光染色，例如DAPI，钙黄绿素AM，或适当的细胞系按照制造商的说明其他细胞跟踪化合物在玻片的介质。 注:对于侵袭的定量，在整个球体均匀染色不是必需的。对于需要染色更深的渗透，较长的孵育应用（&gt; 3小时）可能是必要的。 </li><li>清除污渍，用500微升温暖1X PBS代替。在cubate在37℃下进行10分钟，以除去多余的污渍。重复至少两次。 </li><li>使用荧光显微镜，通过侵入球状体获得光学截面。 注:长期暴露于激光重复应该在成像过程中被最小化，以限制光毒性和光漂白。 </li><li>使用软件如ImageJ的生成的Z突起，其可用于量化球体的总面积，侵入细胞，和距离的数字侵入到胶原基质。 </li><li>作为一个例子，以量化侵袭，调整图像阈排除背景，突出仅球体和侵入细胞，并测量它们的面积。原始球体的面积可以从该总减去量化侵袭。 </li></ol> 5.定量入侵:免疫荧光注:所有的解决方案和缓冲区应通过一个0.45微米的过滤器来传递使用前圣雷莫已经碎片，它可以染色产生不利影响。 <ol><li>准备PBS +洗涤液。制备1×PBS中并加入氯化钙和氯化镁至0.1毫米的最终浓度。 氯化钙和氯化镁加入后不要高压缓冲。溶液可以过滤灭菌，并储存在室温下。 </li><li>制备中性缓冲福尔马林（NBF）固定缓冲区。加入5滴的1M的NaOH至0.6g多聚甲醛。带来至20毫升，用PBS +和在60℃下孵育直至低聚甲醛溶解（约1小时）。冷却至室温并调节pH值至7.4，用1M HCl中。 注:NBF固定缓冲区应立即使用前准备。 </li><li>制备牛血清白蛋白（BSA）封闭缓冲液。在3％w / v的溶解的BSA在PBS +。溶液可以过滤灭菌并且储存于4℃几天，但最好是新鲜制备。不要加热。 </li><li>准备通透缓冲区。稀释的Triton X-100以在PBS + 0.2％体积/体积。 注:通透缓冲区应立即使用前准备。 </li><li> （可选）准备安装MOWIOL中。结合2.4克MOWIOL，6克甘油，和6毫升超纯H 2 O混合在在室温下在旋转器3小时。加入12毫升0.2M的三Cl（pH值为8.5）。与在50℃下混合10分钟孵育。 <ol><li>离心机在5000×g离心15分钟以沉淀不溶物质。添加抗漂白剂，如2.5％DABCO。储存在-20°C 500微升等分。 </li></ol></li><li> 球状体的免疫荧光染色（图5） 注:使用吸管慢慢地从室幻灯片中添加或删除液体。避免使用吸气的，因为这可能破坏胶原层。 <ol><li>准备球状体和嵌入在胶原填充室载玻片，如在章节1中记载为2。 注:胶原蛋白的自发荧光可以通过使用薄层或c的小体积最小化ollagen。 </li><li>从每一个腔室滑动除去尽可能多培养基越好。 </li><li>简单地说用500微升PBS +洗涤液冲洗胶原层以去除残留的网上平台。 </li><li>加入500微升新鲜PBS +的洗涤缓冲液到每个孔中，并在37℃下孵育5分钟以洗涤球粒。重复两次。 </li><li>用500微升NBF固定缓冲液更换PBS +洗涤液。在室温下孵育20 - 25分钟。 </li><li>删除NBF固定缓冲区，并用500微升PBS +洗涤液冲洗。洗5分钟，用新鲜的PBS +洗涤缓冲液至少3次。 注:固定球体可以储存在4℃下在新鲜的PBS +的洗涤缓冲液几天。 0.01％叠氮化钠可以加入到洗涤缓冲液贮存期间，以抑制微生物的生长。 </li><li>更换PBS洗涤+缓冲区500μL通透缓冲区。在室温下孵育10-15分钟。 </li><li><stro纳克&gt;替换透化缓冲液500微升BSA封闭缓冲液并在室温下孵育1小时。 <ol><li>也可以删除BSA封闭液。使用手术刀或剪刀，消费球体和胶原层围绕着三个x 3毫米的区域。使用大口径P1000尖，用新鲜的BSA封闭液轻轻漂洗去除胶原蛋白的切除块。胶原蛋白转移块1.5毫升管。 </li><li>离心2000 XG 2分钟，弃上清。该管可以仔细倒置在干净的纸巾吸走多余的液体。 </li></ol></li><li>初级抗体添加到球状体并在室温下孵育1小时。添加第一抗体的足够体积，以使整个胶原层被均匀地淹没。上述可选步骤（5.6.8.1-5.6.8.2）通常允许使用抗体的小卷。 </li><li> 淹没玻片与至少10毫升的PBS +洗BUF的容器FER 10分钟以洗涤。重复至少两次。 <ol><li>可选，如果球体从以前的胶原层（步骤5.6.8.1）切除，增加至少1毫升的PBS +洗缓冲到1.5毫升管，轻轻地搅动。允许浸泡至少10分钟。 </li><li>除去尽可能多PBS +的洗涤缓冲液尽可能并重复洗涤用PBS +洗涤缓冲至少两次。离心机在2000 XG数分钟沉淀胶原块。 注:通过浸没在洗涤缓冲液前，用70％的乙醇擦拭室滑动的清洁外表面。 </li></ol></li><li>重复步骤5.6.9-5.6.10使用适当的二级抗体。 注:如果球状体在成像器直接染色，继续执行步骤5.6.13。 </li><li> 可选的，如果球状体由胶原层以前切出（步骤5.6.8.1），清洁载玻片并用70％的乙醇共焦显微镜兼容盖玻片。<ol><li>取下胶原块，重悬在超纯H 2尽可能多的洗涤液尽可能O.在安装之前，立即执行此步骤。不要把彩色块浸泡在水中。 </li><li>用细尖镊子，把握胶原块，并转移到载玻片的边缘。 </li><li>使用镊子举行的地方胶原蛋白，用干净的棉签胶原块灯芯多余的液体，确保不要直接触摸的胶原蛋白。 注意:如果胶原卡住到棉签它可以使用镊子轻轻除去，或通过在水中浸没。 </li></ol></li><li> 使用反向移液技术，免除100微升MOWIOL安装介质上的胶原蛋白块，地点在盖玻片样品。 <ol><li>使用棉签或纸巾从盖玻片下灯芯过剩MOWIOL封固剂和水了。 </li><li>允许MOWIOL安装介质在4℃过夜硬化盖玻片C.封边用指甲油。 </li></ol></li><li>利用共聚焦或荧光显微镜图像染色球体观察的兴趣，形成侵入结构等 （ 图5B，5C）蛋白质的定位。 注:彩色球体应加以保护，以防止漂白。 </li></ol></li></ol> 6.失巢凋亡分析注:球体形成协议很容易适应量化锚定非依赖性生长并失巢凋亡抗性的各种细胞类型。 <ol><li> 接种细胞失巢凋亡测定（图6） <ol><li>遵循球体生产指令1.4节，但至少使用30毫克/毫升甲基纤维素制备球状体形成网上平台。 注:当回温，30毫克/毫升甲基纤维素应该产生一个厚厚的凝胶保存细胞悬浮液中。 </li><li>孵育几天细胞周和在在用显微镜放大10倍定期SPECT。能抵抗失巢凋亡细胞应增殖并形成菌落。 </li><li>通过测量形成在每个孔集落的数目和大小量化失巢凋亡。 注:一旦菌落形成的，它们可以被洗涤以除去甲基纤维素和用于基于球状体的测定法，如所描述。 </li></ol></li></ol>

<ol>
	<li>Zimmermann, M., Box, C., Eccles, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+vs.+three-dimensional+in+vitro+tumor+migration+and+invasion+assays.">Two-dimensional vs. three-dimensional in vitro tumor migration and invasion assays.</a> Methods Mol Biol. 986 (986), 227-252 (2013).</li><li>Albini, A., Noonan, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'chemoinvasion'+assay,+25+years+and+still+going+strong:+the+use+of+reconstituted+basement+membranes+to+study+cell+invasion+and+angiogenesis.">The 'chemoinvasion' assay, 25 years and still going strong: the use of reconstituted basement membranes to study cell invasion and angiogenesis.</a> Curr Opin Cell Biol. 22 (5), 677-689 (2010).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nat. Rev. Mol. Cell Biol. 8 (10), 839-845 (2007).</li><li>Fennema, E., Rivron, N., Rouwkema, J., van Blitterswijk, C., de Boer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+culture+as+a+tool+for+creating+3D+complex+tissues.">Spheroid culture as a tool for creating 3D complex tissues.</a> Trends Biotechnol. 31 (2), 108-115 (2013).</li><li>Carletti, E., Motta, A., Migliaresi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+for+tissue+engineering+and+3D+cell+culture.">Scaffolds for tissue engineering and 3D cell culture.</a> Methods Mol Biol. 695, 17-39 (2011).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+hanging+drop+cell+culture+protocol+for+generation+of+3D+spheroids.">A simple hanging drop cell culture protocol for generation of 3D spheroids.</a> J Vis Exp. (51), (2011).</li><li>Handschel, J. G., 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=Prospects+of+micromass+culture+technology+in+tissue+engineering.">Prospects of micromass culture technology in tissue engineering.</a> Head Face Med. 3, 4 (2007).</li><li>Stuckhoff, A. P., DelValle, L., Amini, S., White, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuronal Cell Culture Methods and Protocols Vol. 1078 Methods in Molecular Biology. 1078, 119-132 (2013).</li><li>Stewart, G. J., Wang, Y., Niewiarowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylcellulose+protects+the+ability+of+anchorage-dependent+cells+to+adhere+following+isolation+and+holding+in+suspension.">Methylcellulose protects the ability of anchorage-dependent cells to adhere following isolation and holding in suspension.</a> Biotechniques. 19 (4), 598-604 (1995).</li><li>Vinci, 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=Advances+in+establishment+and+analysis+of+three-dimensional+tumor+spheroid-based+functional+assays+for+target+validation+and+drug+evaluation.">Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation.</a> BMC Biol. 10, 29 (2012).</li><li>Nagelkerke, A., Bussink, J., Sweep, F. C. G. J., Span, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+multicellular+tumor+spheroids+of+breast+cancer+cells:+How+to+go+three-dimensional.">Generation of multicellular tumor spheroids of breast cancer cells: How to go three-dimensional.</a> Anal. Biochem. 437 (1), 17-19 (2013).</li></ol>

The authors have nothing to disclose.

我们提出了生产3D细胞球体使用廉价的和广泛使用的试剂在体内组织的架构模型快速，灵活的方法。我们的协议利用MC 8,9的非细胞毒性和粘合促进性质介导的细胞聚集和降低细胞单层的形成。不像从动物来源分离的基于蛋白质的基质，MC是惰性的，不包含生长因子，并且很容易通过洗涤除去，允许球状体在各种应用中使用的隔离无残余基质的存在?...

的肿瘤细胞的行为生物相关的评估是使用传统的二维（2D）细胞培养的方法，部分是因为这些不充分地反映在体内发现的细胞微环境挑战。结合细胞外基质成分进入文化（ 如 Boyden小室测定）的替代方法是体内组织的环境更生理代表。然而，它们可被限定在单个细胞行为的评估，并且不概括在有助于组织或肿瘤生长1，2，3细胞-基质和细胞-细胞相互作用的体内组合的复合物。 使用多细胞球体是最近的做法更准确地再现体内细胞生长1的结构紧凑，4。球状体可以用于研究正常细胞的细胞-基质相互作用，而且还可以作为肿瘤类似物肿瘤进展的特征，如转移性生长或药物抗性4建模。 球状体可以通过嵌入在基体5，或更快速的单细胞的增殖而形成，通过促进多种细胞的聚集，以形成一单细胞群（ 例如 ，悬滴，离心方法）6,7。现有的细胞聚集技术可能需要昂贵的材料或专门设备。此外，这些球状体具有宽范围的尺寸和形态，并且可以是难以生产大批量，使得生长条件或处理困难之间的比较。最后，通过这些方法产生的球状体可能难以从蛋白质extracel隔离细胞性矩阵，其中它们嵌入在其他应用中使用。 在这里，我们描述了用于快速形成使用市售U型底细胞斥板和惰性粘合促进基质，甲基纤维素一致大小细胞球体的一个强大的和易于修改细胞聚集方法。一旦形成，这些多细胞球状体可容易地分离出一种用于在广泛的应用中使用。该协议也容易适于产生通过细胞增殖，其可用于评估其他细胞过程球状体。这里，我们表明细胞侵袭测定法，免疫荧光染色定量，一个失巢凋亡测定中，由于这两种不同的球体形成的协议的应用实例。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x Phosphate buffered saline</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Solution</td>
			<td>Sigma-Aldrich</td>
			<td>21114</td>
			<td>Used for PBS* wash buffer; Do not autoclave PBS* wash buffer upon addition of calcium chloride</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Solution</td>
			<td>Sigma-Aldrich</td>
			<td>M1028</td>
			<td>Used for PBS* wash buffer; Do not autoclave PBS* wash buffer upon addition of magnesium chloride</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>For Spheroid Formation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well U-bottom Cell-Repellent Plate</td>
			<td>Greiner Bio-One</td>
			<td>650970</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Sigma-Aldrich</td>
			<td>D5546</td>
			<td>For culturing SH-SY5Y, PANC-1, TPC-1 cell lines</td>
		</tr>
		<tr>
			<td>F12K Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>2112722</td>
			<td>For culturing TT cell line</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F1051</td>
			<td>Filter prior to use to remove particulate contaminants</td>
		</tr>
		<tr>
			<td>Methyl cellulose</td>
			<td>Sigma-Aldrich</td>
			<td>M7027</td>
			<td>Prepare in water to 100 mg/mL</td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute Medium</td>
			<td>Sigma-Aldrich</td>
			<td>R8758</td>
			<td>For culturing HCT-116, BxPC-3 cell lines</td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605028</td>
			<td>Dissociation buffer</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>For Invasion Assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Type I Collagen</td>
			<td>Corning Incorporated</td>
			<td>354231</td>
			<td>Stock 3.1mg/ml; Maintain on ice when in use</td>
		</tr>
		<tr>
			<td>DMEM Phenol Red Free Low Glucose&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>11054-20</td>
			<td>Less background fluorescence compared to Phenol Red supplemented medium</td>
		</tr>
		<tr>
			<td>Glial Cell Line Derived Neurotrophic Factor</td>
			<td>Peprotech</td>
			<td>450-10</td>
			<td>Chemoattractant</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>For Immunofluorescence Microscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>#1.5 Coverglass</td>
			<td>Electron Microscopy Sciences</td>
			<td>72225-01</td>
			<td>For mounting excised spheroids</td>
		</tr>
		<tr>
			<td>Alexa-Fluor 488 Phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12379</td>
			<td>Used to stain actin at 1:200</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Bioshop Canada Incorporated</td>
			<td>ALB001</td>
			<td>Used in BSA blocking buffer</td>
		</tr>
		<tr>
			<td>Dabco 33-LV</td>
			<td>Sigma-Aldrich</td>
			<td>290734</td>
			<td>Antifade</td>
		</tr>
		<tr>
			<td>Glycerol&#160;</td>
			<td>Bioshop Canada Incorporated</td>
			<td>GLY001</td>
			<td>Used in MOWIOL mounting medium</td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>Freeware, NIH</td>
			<td>-</td>
			<td>Used for image analysis&#160;</td>
		</tr>
		<tr>
			<td>Microslides</td>
			<td>VWR International</td>
			<td>48312-024</td>
			<td>For mounting excised spheroids</td>
		</tr>
		<tr>
			<td>MOWIOL 4-88</td>
			<td>EMD-Millipore</td>
			<td>475904</td>
			<td>Used in MOWIOL mounting medium</td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>EMD-Millipore</td>
			<td>PX0055-3</td>
			<td>Used in fixation buffer</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Bioshop Canada Incorporated</td>
			<td>TRX777</td>
			<td>Used in permeabilization buffer</td>
		</tr>
	</tbody>
</table>

an efficient and flexible cell aggregation method for 3d spheroid production

单层细胞培养不充分模拟组织的体内行为，这涉及复杂的细胞-细胞和细胞-基质相互作用。三维（3D）细胞培养技术是最近开发用于解决贴壁细胞培养的缺点一个创新。而在体外产生组织类似物几种技术已经开发，这些方法往往是复杂，以建立价格昂贵，需要专门的设备，并且通常由兼容性只有某些细胞类型的限制。在这里，我们描述了用于凝集细胞成一致的大小是与多种肿瘤和正常细胞系的生长兼容的多细胞三维球状体一个快速和灵活的协议。我们利用不同浓度的血清和甲基纤维素（MC），以促进依赖贴壁球体的产生和防止在一个高度可再生的方式的细胞单层的形成。逐张为最佳条件idual细胞系可以通过在球体形成介质调整MC或血清浓度来实现。生成的3D球体可被收集用于在广泛的应用范围，包括细胞信号传导或基因表达研究中，候选药物筛选，或在细胞过程如肿瘤细胞侵袭和迁移的研究中使用。该协议也容易地适用于产生从单个细胞克隆的球状体，并可以适于评估贴壁依赖性生长和失巢凋亡抗性。总的来说，我们的协议提供了用于产生和利用为了概括组织的微环境的3D模型和正常和肿瘤细胞的体内生长三维细胞球状体的容易修改的方法。

我们描述一个灵活，高效的方法来生成利用细胞排斥板和补充MC球体形成媒体离散球体。下MC和血清的适当的条件下，各个细胞沉降并在井的中心粘附在一起以形成具有最小粘附到孔底部的球体。使用该协议，是从多种细胞系中（ 图2B）产生球体。 MC和血清浓度的滴定所需的每个细胞系，以确定在形成仅单个球体即足够强大，以允许操纵未经分段的最佳条件。?...

Watch this Scientific Journal Video about An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production at JoVE.com

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

三维球体制作一个高效，灵活细胞聚集方法

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

an-efficient-flexible-cell-aggregation-method-for-3d-spheroid

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

23.6K 次观看.  Queen's University Cancer Research Institute. Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions. 

视频: An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

科研

生物学

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法<em&gt;拟南芥</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

一种有效的体外转位方法由转录调节的睡眠美容系统封装到一个集成缺陷的慢向量

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

共聚焦显微镜通过图像相关谱揭示细胞表面受体聚集

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users&#8217; needs.

Presented here is a protocol for the fabrication of a spheroid imaging device. This device enables dynamic or longitudinal fluorescence imaging of cancer cell spheroids. The protocol also offers a simple image processing procedure for the analysis of cancer cell invasion.

3D球形模型中癌细胞入侵的时限解荧光成像和分析

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

一种简单灵活的接种方法，用于准确评估拟南芥和其他植物的白粉病感染表型

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

使用荧光相关光谱检测蛋白质聚合

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

拟南芥种子表面灭菌的高通量、稳健性和高时间灵活性方法

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental ...

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale quantitative phenotypic measurements from cells. This approach has proved powerful for deepening our understanding of a wide range of both fundamental and applied events in cell biology. To date, the majority of applications for this technology have relied on the use of cells grown in monolayers, although it is increasingly realized that such models do not recapitulate many of the interactions and processes that occur in tissues. As such, there has been an emergence in the development and use of 3-dimensional (3D) cell assemblies, such as spheroids and organoids. Although these 3D models are particularly powerful in the context of cancer biology and drug delivery studies, their production and analysis in a reproducible manner suitable for HCS and HCA present a number of challenges. The protocol detailed here describes a method for the generation of multicellular tumor spheroids (MCTS), and demonstrates that it can be applied to three different cell lines in a manner that is compatible with HCS and HCA. The method facilitates the production of several hundred spheroids per well, providing the specific advantage that when used in a screening regime, data can be obtained from several hundred structures per well, all treated in an identical manner. Examples are also provided, which detail how to process the spheroids for high-resolution fluorescence imaging and how HCA can extract quantitative features at both the spheroid level as well as from individual cells within each spheroid. This protocol could easily be applied to answer a wide range of important questions in cell biology.

This protocol details a method for the production of three different types of spheroids in a manner that makes them suitable for large-scale high-content screening and analysis. In addition, examples are presented showing how they can be analyzed at spheroid and individual cell levels.

用于高内涵筛选和分析应用的大规模生产球体的稳健方法

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale ...

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen infection or challenge with pathogen-associated chemicals (pathogen-associated molecular patterns [PAMPs]), an array of immune responses, including a ROS burst, are quickly induced in plants, which is called PAMP-triggered immunity (PTI). A ROS burst is a hallmark PTI response, which is catalyzed by a group of plasma membrane-localized NADPH oxidases-the RBOH family proteins. The vast majority of ROS comprise hydrogen peroxide (H2O2), which can be easily and steadily detected by a luminol-based chemiluminescence method. Chemiluminescence is a photon-producing reaction in which luminol, or its derivative (such as L-012), undergoes a redox reaction with ROS under the action of a catalyst. This paper describes an optimized L-012-based chemiluminescence method to detect apoplast ROS production in real-time upon PAMP elicitation in rice tissues. The method is easy, steady, standardized, and highly reproducible under firmly controlled conditions.

Here, we describe a method for the real-time detection of apoplastic reactive oxygen species (ROS) production in rice tissues in pathogen-associated molecular pattern-triggered immune response. This method is simple, standardized, and generates highly reproducible results under controlled conditions.

用化学发光法实时检测水稻免疫反应中活性氧的产生

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen ...