这项研究由国家科学基金会奖1237969号资助, "解开马铃薯的杂合性, 等位成分, 和拷贝数变异" 和美国农业部特别赠款 2014-34141-22266 (缅因州大学) 到 RV。

注意: 必须包括适当的控制, 通常以与实验样品相同倍体的叶组织形式。这是因为块茎样品的2C 峰值可能很小, 很难辨认。
1. 筹备工作
注意: 此处列出的解决方案可能提前准备好, 并存储在4摄氏度, 但在使用当天必须将其放在后续步骤中。
<ol><li>制作适当数量的质壁分离孵化解决方案 (pi) (15 毫升/样本): 0.55 米甘露醇, 2 毫米CaCl2, 1 毫米 2 PO 4, 1 毫米氯化镁 2, 50 毫米三缓冲 pH 值 7.5 (与 HCl 调整).</li>
	<li>制作适当体积的质壁分离洗涤液 (PWS) (15 毫升/样品), 这是相同的 pi 除了增加0.71 米甘露醇。</li>
	<li>制作250毫升的改良的 FCB 流式细胞术缓冲器11 (): 13.6 毫米柠檬酸钠 (三钠), 8 毫米拖把, 18 毫米氯化镁 2, 0.4% v/v 海卫 X-100.搅拌至少30分钟, 以分发海卫 X-100。</li>
	<li>过滤器消毒 pi 和 PWS 解决方案与0.22 µm 不对称聚醚砜 (猿) 过滤器。使用真空驱动过滤装置。</li>
	<li>
为裂解原生质体准备106µm 滤网过滤器。准备1.5 毫升离心管, 可直接嵌套在收集管, 但任何通过106µm 过滤器通过悬浮的方法可以使用。 注意: 这些不需要是无菌的。<ol>
<li>如果使用1.5 毫升离心管, 切断1厘米的尖端每离心管。使用热板或其他热源加热1厘米2片106µm 钢网在一块铝箔上。将管子的切口端压入网格, 直到熔化。</li>
	</ol>
</li>
	<li>洗土豆被取样, 清除所有的土壤和碎片。 注: 块茎大小和年龄应适合实验的目的, 但似乎不影响质量的结果。我们已经成功地将此协议应用于已在冷藏 (4 摄氏度, 95% RH) 的块茎, 直到8月。虽然块茎有缺陷 (例如块茎末端腐烂, 空心心脏, 褐色坏死区) 可能反应, 他们应该避免, 如果可能的话。</li>
</ol>2. 1 天: Plasmolyze 块茎组织
注: 控制叶样品可以在这里包括生产原生质体或在步骤 5.2, 如果原油的准备是首选。这必须用无菌工具在无菌环境中进行, 如层流罩。
<ol><li>通过浸泡75% 乙醇, 对马铃薯块茎进行表面杀菌, 至少5分钟。</li>
	<li>从乙醇和空气干燥中除去块茎。这通常需要5分钟, 更长的时间, 如果块茎大于100克。</li>
	<li>准备和标签无菌50毫升圆锥管为每个样品和整除15毫升过滤消毒的 pi 到每个。</li>
	<li>
使用灭菌的手术刀或软木塞, 从灭菌的块茎中取样大约1厘米3的所需组织。 注: 根据取样的组织, 可以使用灭菌的软木塞或刀/手术刀。例如, 如果需要髓核, 软木钻可以用来从顶端到块茎远端的核心, 可以取样核心部分。然而, 由于马铃薯块茎的内部形态不对称, 通过将块茎减半并切除所需的组织, 可以更精确地取样实质或皮质。有关内部块茎形态学的描述, 请参见图 1 。<ol>
<li>粗切组织使用消毒手术刀到大约3毫米3件和转移组织到圆锥管包含 pi。孵化过夜在4摄氏度。 注: 这是为了最大化表面积, 使 pi, 后来的酶解, 可能充分渗透组织导致更彻底的消化。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57134/57134fig1.jpg"> 图 1: 马铃薯块茎的内部形态.用黑线说明了髓 (P) 与 perimedullary 实质 (PM) 的分离。从皮层 (C) 分离实质的血管环用黑色箭头表示。stolon 端 (S) 和块茎眼睛 (E) 也被标记。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57134/57134fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
3. 2 天: 产生马铃薯原生质体
注意: 这必须用无菌工具在无菌环境中进行, 如层流罩。
<ol><li>准备和过滤 (0.45 µm 猿过滤器) 适当数量的酵素解答 (ES) (10 毫升/样品): 0.71 米甘露醇, 3 毫米 CaCl2, 1 毫米.2PO4, 1 毫米氯化镁2, 4% Onozuka R-10 纤维素酶, 0.8% 离析酶 R-10, 1% hemicululase, 105.8。确保此步骤中使用了适当的筛选器;孔径小于0.45 µm 的过滤器容易堵塞, 而低蛋白结合膜 (如猿类) 可最大限度减少过滤过程中酶的损失。</li>
	<li>用不育的血清吸管从圆锥管中的样品中吸取 pi。</li>
	<li>在每个样品中加入10毫升的 ES, 并反转2–3倍。</li>
	<li>孵化样品隔夜在29°c 与 180 rpm 水平震动。孵育样品至少16小时。</li>
</ol>4. 3 天: 收获原生质体和洗涤原生质体
注: 无菌条件在这一点上是不必要的。
<ol><li>从振动筛中取出样品, 并允许它们沉降10分钟。</li>
	<li>
尽可能多地吸取 ES 解决方案。
	<ol>
<li>用血清学吸管去除尽可能多的 ES 溶液, 注意避免去除被消化的组织。</li>
		<li>使用微删除任何剩余 ES 溶液, 再次避免消化组织。这是最容易做到通过持有管在一个角度, 使 ES 溶液流向盖子, 而消化组织保持在底部。</li>
	</ol>
</li>
	<li>在每个样品中加入15毫升的 PWS, 并轻轻2–3时间反转。允许原生质体安定10分钟。</li>
	<li>用血清学吸管去除 PWS。使用微删除任何剩余的液体。 注意: 在流式细胞术过程中, 这一步骤绝对是确保样本核完整性的关键。在疑难解答中, 此步骤被发现是示例中最可能出现故障的原因。</li>
</ol>5. 3 天: 为流式细胞术准备样品
注: 除非另行注明, 样品应在冰上保存。
<ol><li>碘碘 (0.4 毫克碘碘化物/毫升 FCB) 和 RNase 溶液 (0.8 毫克 RNase/毫升 FCB), 通过溶解在 FCB。 注意: 碘碘化物具有剧毒。避免接触皮肤或眼睛。穿戴合适的 PPE。</li>
	<li>
在每个样品中加入1.5 毫升的冰冷 FCB。简单地摇动或涡旋每个样本以分解聚合组织。重要的是要打破块茎组织的团簇, 使 FCB 可以充分渗透细胞和释放细胞核。不同的样品可能需要或多或少的搅拌取决于组织和基因型。 注: FCB 裂解原生质体, 释放细胞核。因此, 必须在冰上保存样品以防止降解。<ol>
<li>如果控制流式细胞术样本不包括在原生质体生成步骤, 他们可能会包括在这里。如果要添加控件, 请用剃刀刀片在1.5 毫升的冰冷 FCB 中用刀片细砍一小片叶子 (~ 3 厘米2)。对照样品应与实验样品具有相同的倍性, 最好是相同的基因型。体外试管苗比温室或田间种植的植物更容易提供更清洁的峰值。</li>
	</ol>
</li>
	<li>
通过106µm 网格过滤器通过1毫升的 FCB/组织悬浮液。使用1.5 毫升离心管与尖端切断和金属网熔化到底部, 如步骤1.5.1 所述。此离心管可直接嵌套在2毫升离心管中, 样品直接通过。如果使用另一种过滤方法, 滤液可以收集在冰冷的培养板, 然后转移到2毫升管。 注: 有时剩余的骨料和细胞碎片可能堵塞网格过滤器。在这种情况下, 只需轻敲两个嵌套的离心管几次就可以去除碎片。</li>
	<li>将 RNase 解决方案的250µL 添加到每个示例中。室温下反转和孵育30分钟。 注意: 此步骤旨在从样本中去除 RNA, 从而在流式细胞术中减少噪音。然而, 由于细胞核是短命的, 研究人员可能会决定减少孵化时间或在冰上执行, 如果他们正在经历严重退化的样品。</li>
	<li>将碘碘化液的125µL 添加到每个样品中。在冰上反转和孵化30分钟。 注: 样品应尽快用于流式细胞术, 因为降解是明显的在2小时内, 即使在冰上。</li>
</ol>6. 3 天: 马铃薯块茎核流式细胞术
注: 流式细胞仪的操作和软件将根据仪器和制造商的不同而有所不同。关于流式细胞仪的具体操作的详细说明将在仪器的用户指南中提供。
<ol><li>创建两个点图, 使用对数尺度的正散射与侧向散射和碘碘 (PI) vs 侧向散射。还可以在 x 轴上创建一个带有 PI 的直方图。对数尺度是必需的, 以确保所有的事件都在规模内, 因为原子核可能大大不同的荧光。</li>
	<li>
加载已知的控制采样管并调整电压以使所有事件都处于刻度。我们使用体外叶组织的样本基因型。如果要运行不同 ploidies 的样本, 应包括每个控件的样本。
	<ol>
<li>在控制样品中记下2C 峰值的通道。实验样品的2C 峰值应落在同一位置。</li>
	</ol>
</li>
	<li>加载一个实验 (块茎) 样本, 并再次确保所有事件的规模。如果需要进行调整, 请重复步骤6.2 以确定2C 峰值的通道。</li>
	<li>人工门的原生质体核使用侧散射 vs PI 情节。</li>
	<li>设置 PI 直方图只显示门控原生质体核区域。</li>
	<li>从每个示例中收集所需的事件数。研究人员经常使用1万门控事件进行流式细胞术;然而, 我们使用2000事件的块茎原生质体样本, 以容纳更多的样本和低浓度样品。</li>
	<li>使用以下公式从 PI 直方图中计算每个样本的 EI: EI =<img alt="Equation 1" src="/files/ftp_upload/57134/57134eq1.jpg"> 其中4C 是 4C (代表一轮 endoreduplication) 的原子核百分比, 8C 是8C 的百分比, 等等。有关顶点的直方图和 C 值的示例, 请参见图 4 。</li>
</ol>

<ol>
	<li>Wilson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The karyoplasmic ratio. 727, (1925).</li><li>Szymanski, D. B., Marks, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GLABROUS1+overexpression+and+TRIPTYCHON+alter+the+cell+cycle+and+trichome+cell+fate+in+Arabidopsis.">GLABROUS1 overexpression and TRIPTYCHON alter the cell cycle and trichome cell fate in Arabidopsis.</a> Plant Cell. 10 (12), 2047-2062 (1998).</li><li>Melaragno, J. E., Mehrotra, B., Coleman, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+endopolyploidy+and+cell+size+in+epidermal+tissue+of+Arabidopsis.">Relationship between endopolyploidy and cell size in epidermal tissue of Arabidopsis.</a> Plant Cell. 5 (11), 1661-1668 (1993).</li><li>Grafi, G., Larkins, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoreduplication+in+maize+endosperm:+involvement+of+M+phase--promoting+factor+inhibition+and+induction+of+S+phase--related+kinases.">Endoreduplication in maize endosperm: involvement of M phase--promoting factor inhibition and induction of S phase--related kinases.</a> Science. 269 (5228), 1262-1264 (1995).</li><li>Schweizer, L., Yerk-Davis, G., Phillips, R., Srienc, F., Jones, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+maize+endosperm+development+and+DNA+endoreduplication.">Dynamics of maize endosperm development and DNA endoreduplication.</a> Proc Natl Acad Sci. 92 (15), 7070-7074 (1995).</li><li>Chen, C. T., Setter, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+potato+tuber+cell+division+and+growth+to+shade+and+elevated+CO2.">Response of potato tuber cell division and growth to shade and elevated CO2.</a> Ann Bot. 91 (3), 373-381 (2003).</li><li>Chen, C. T., Setter, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+potato+dry+matter+assimilation+and+partitioning+to+elevated+CO2+at+various+stages+of+tuber+initiation+and+growth.">Response of potato dry matter assimilation and partitioning to elevated CO2 at various stages of tuber initiation and growth.</a> Environ Exp Bot. 80, 27-34 (2012).</li><li>Cheniclet, C., 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=Cell+expansion+and+endoreduplication+show+a+large+genetic+variability+in+pericarp+and+contribute+strongly+to+tomato+fruit+growth.">Cell expansion and endoreduplication show a large genetic variability in pericarp and contribute strongly to tomato fruit growth.</a> Plant Physiol. 139 (4), 1984-1994 (2005).</li><li>Pirrello, J., 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=How+fruit+developmental+biology+makes+use+of+flow+cytometry+approaches.">How fruit developmental biology makes use of flow cytometry approaches.</a> Cytometry Part A. 85 (2), 115-125 (2014).</li><li>Chevalier, C., 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=Endoreduplication+and+fruit+growth+in+tomato:+evidence+in+favour+of+the+karyoplasmic+ratio+theory.">Endoreduplication and fruit growth in tomato: evidence in favour of the karyoplasmic ratio theory.</a> J Exp Bot. 65 (10), 2731-2746 (2014).</li><li>Galbraith, D. W., 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=Rapid+flow+cytometric+analysis+of+the+cell+cycle+in+intact+plant+tissues.">Rapid flow cytometric analysis of the cell cycle in intact plant tissues.</a> Science. 220 (4601), 1049-1051 (1983).</li><li>Dole&#382;el, J., Greilhuber, J., Suda, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+Cytometry+with+Plants:+An+Overview.">Flow Cytometry with Plants: An Overview.</a> Flow Cytometry with Plant Cells. , 41-65 (2007).</li><li>Dole&#382;el, J., Barto&#353;, J. A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+DNA+flow+cytometry+and+estimation+of+nuclear+genome+size.">Plant DNA flow cytometry and estimation of nuclear genome size.</a> Ann Bot. 95 (1), 99-110 (2005).</li><li>Doke, N., Tomiyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hyphal+wall+components+from+Phytophthora+infestans+on+protoplasts+of+potato+tuber+tissues.">Effect of hyphal wall components from Phytophthora infestans on protoplasts of potato tuber tissues.</a> Phys Plant Path. 16 (2), 169-176 (1980).</li><li>Davis, D. A., Currier, 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=The+effect+of+the+phytoalexin+elicitors,+arachidonic+and+eicosapentaenoic+acids,+and+other+unsaturated+fatty+acids+on+potato+tuber+protoplasts.">The effect of the phytoalexin elicitors, arachidonic and eicosapentaenoic acids, and other unsaturated fatty acids on potato tuber protoplasts.</a> Physiol Mol Plant Pathol. 28 (3), 431-441 (1986).</li><li>Laimbeer, F. P. 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=Protoplast+isolation+prior+to+flow+cytometry+reveals+clear+patterns+of+endoreduplication+in+potato+tubers,+related+species,+and+some+starchy+root+crops.">Protoplast isolation prior to flow cytometry reveals clear patterns of endoreduplication in potato tubers, related species, and some starchy root crops.</a> Plant Methods. 13 (1), 27 (2017).</li><li>Uijtewaal, B. A., Huigen, D. J., Hermsen, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+potato+monohaploids+(2n=x=12)+through+prickle+pollination.">Production of potato monohaploids (2n=x=12) through prickle pollination.</a> Theor Appl Genet. 73 (5), 751-758 (1987).</li><li>Karp, A., Risiott, R., Jones, M. G. K., Bright, S. W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+doubling+in+monohaploid+and+dihaploid+potatoes+by+regeneration+from+cultured+leaf+explants.">Chromosome doubling in monohaploid and dihaploid potatoes by regeneration from cultured leaf explants.</a> Plant Cell Tiss Org Cult. 3 (4), 363-373 (1984).</li><li>Doke, 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+superoxide+anion+by+potato+tuber+protoplasts+during+the+hypersensitive+response+to+hyphal+wall+components+of+Phytophthora+infestans+and+specific+inhibition+of+the+reaction+by+suppressors+of+hypersensitivity.">Generation of superoxide anion by potato tuber protoplasts during the hypersensitive response to hyphal wall components of Phytophthora infestans and specific inhibition of the reaction by suppressors of hypersensitivity.</a> Phys Plant Path. 23 (3), 359-367 (1983).</li><li>Doke, N., Tomiyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+the+hypersensitive+response+of+potato+tuber+protoplasts+to+hyphal+wall+components+by+water+soluble+glucans+isolated+from+Phytophthora+infestans.">Suppression of the hypersensitive response of potato tuber protoplasts to hyphal wall components by water soluble glucans isolated from Phytophthora infestans.</a> Phys Plant Path. 16 (2), 177-186 (1980).</li><li>Saxena, P. K., King, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reuse+of+enzymes+for+isolation+of+protoplasts.">Reuse of enzymes for isolation of protoplasts.</a> Plant Cell Rep. 4 (6), 319-320 (1985).</li><li>Sgorbati, S., Levi, M., Sparvoli, E., Trezzi, F., Lucchini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytometry+and+flow+cytometry+of+4&#8242;,6-diamidino-2-phenylindole+(DAPI)-stained+suspensions+of+nuclei+released+from+fresh+and+fixed+tissues+of+plants.">Cytometry and flow cytometry of 4&#8242;,6-diamidino-2-phenylindole (DAPI)-stained suspensions of nuclei released from fresh and fixed tissues of plants.</a> Physiol Plant. 68 (3), 471-476 (1986).</li><li>Bhojwani, S. S., Dantu, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+and+cell+culture.">Tissue and cell culture.</a> Plant Tissue Culture: An Introductory Text. , 39-50 (2013).</li><li>Flores, H. E., Kaur-Sawhney, R., Galston, A. W., Maramorosch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protoplasts+as+Vehicles+for+Plant+Propagation+and+Improvement.">Protoplasts as Vehicles for Plant Propagation and Improvement.</a> Advances in Cell Culture. 1, 241-279 (1981).</li><li>Reeve, R. M., Timm, H., Weaver, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parenchyma+cell+growth+in+potato+tubers+I.+Different+tuber+regions.">Parenchyma cell growth in potato tubers I. Different tuber regions.</a> Am Potato J. 50 (2), 49-57 (1973).</li><li>Scholes, D. R., Paige, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+in+ploidy:+a+generalized+response+to+stress.">Plasticity in ploidy: a generalized response to stress.</a> Trends Plant Sci. 20 (3), 165-175 (2015).</li></ol>

作者声明他们没有竞争的财政利益。

本文提出的协议为研究人员提供了一种评估马铃薯块茎内 endoreduplication 的方法, 其修饰的细胞含量和增加的细胞大小似乎排除了其他流式细胞术的准备。该议定书依赖于原生质体的产生, 以此来减少噪音和碎片, 同时保持核完整。以前, 研究人员已经描述了类似的准备, 特别是顽固流式细胞仪样本, 以及利用块茎原生质体研究各种主题, 如发病机制19,20。然而, 根据我们的知识, 没有人将这种块茎原生质体与流式细胞术结合使用, 以研究 endoreduplication。此外, 我们发现, 原生质体的使用比典型的原油制剂更可靠, 以及用于评估块茎 endoreduplication6,7的其他两项研究中采用的技术。在这里, 我们讨论了协议的缺点, 它的执行和样本准备的潜在缺陷, 以及使用它的典型实验的结果。
尽管块茎流式细胞术协议的实用性和可重复性, 但它确实有一些弱点, 应该讨论。首先, 该协议是时间密集型的, 需要两个通宵孵化。此外, 该议定书需要一些昂贵的试剂, 特别是纤维素酶和离析酶。此外, 准备工作是高度时间敏感的, 在几个小时内降级, 这可能会限制在一天内的吞吐量, 特别是如果需要许多事件。最后, 该议定书虽然可靠, 但对样品制备过程中的误差很敏感, 所有这些都似乎对原子核造成损伤, 并降低了质量结果。例如, 微生物污染可能发生在质壁分离和原生质体生成步骤 (1–3.4) 由于不适当的无菌技术。样本污染, 虽然并不总是排除样品的成功, 但似乎大大降低了获得的直方图的质量, 再次可能是由于对细胞核的破坏。
如前所述, 该协议的主要缺点是成本、时间和对错误的敏感性。研究人员可能希望采取措施来缓解这些问题。就成本而言, 打算将该协议应用于许多样本的研究人员可能会考虑修改酶浓度和/或消化步骤的持续时间, 因为不同的组织和基因型可能因反应而异。这包括筛选和回收以前已演示但在此处未使用21的 ES 的可能性。至于时间, 在我们观察中, 有可能免除第一孵化 (质壁分离步骤), 仍然提取原生质体;然而, 它们的完整性和丰度将受到影响, 这对结果产生不利的后果。为了减少样本的恶化, 研究人员可能会认为一些流式细胞术方法涉及使用护 (例如,甲醛) 来保留样本完整性22。这可能是一个有用的手段, 既防止恶化, 并允许样本存储, 而不是原生质体提取和流式细胞术发生在同一天。防止细胞核损耗的另一种方法可能是将核酸酶抑制剂包括在 ES 和/或 FBC 中;虽然 2-巯基乙醇在发展过程中没有明显的变化, 其他抑制剂还没有在这里测试。
在我们的观察中, 最关键的一步是步骤 4.4, 去除所有质壁分离洗液之前添加流式细胞仪缓冲 (FCB)。如果即使是小体积 (< 10 µL) 仍然存在, 样本更可能被降级, 这可能导致一个贫穷甚至失败的样本。这很可能是由于酶溶液中的杂质(例如核酸, 蛋白酶)23,24 , 在潜伏期和样品运行之间的时间, 即使在低浓度下迅速降解细胞核。另一个重要的考虑因素是 PI 和 RNase 孵化步骤的持续时间。正如《议定书》所指出的, 在添加 FCB 之后, 样品在几个小时内不会保持稳定, 因此研究人员可能决定减少这些步骤的持续时间, 以容纳更多的样本或由低核引起的冗长流式细胞术的运行。浓度.这也要求研究人员考虑在每个样本的事件数和要运行的样本总数之间的权衡, 或者考虑前面提到的护的使用情况。
组织与基因型的差异
为提供该协议的结果代表, 我们设计了两个简单的实验, 以确认以前报告的变化的组织和检查的影响, 倍性和基因型。组织实验的结果表明, 该协议产生可重现的结果, 因为髓组织再次被发现具有最高的 EI。有些令人惊讶的软组织, 以前没有被评估过, 它的 EI 值类似于皮质组织, 明显低于髓。这是意想不到的, 因为软组织细胞通常大于髓或皮质细胞, 至少在成熟。一个可能的解释是, 块茎 (90–130 g) 是太不成熟的薄壁细胞, 其中包括大多数块茎体积成熟, 已完全扩大, 并达到其最大 C 值25。这也可以解释为什么双单倍体 (VT_SUP_19) 和加倍的双单倍体 (VT_SUP_19 4x) 显示出显著大于 cv。当从每个基因型取样大约相等大小的块茎时, 从 VT_SUP_19 或等基因系二、四倍体杂交的块茎的最大大小远小于祖。因此, 可能是因为它们相对于最大可达到的尺寸更大而显示出更大的 EI。或者, 差异可能是遗传补充 VT_SUP_19 从其二、四倍体杂交祖的结果。另一种可能性是, 在从二、四倍体杂交提取双单倍体时发生的基因组减少可能会使有害的等位基因被揭穿, 导致植物范围内的压力, 这也被证明有助于提升的EI 26。然而, 这表明研究人员在选择块茎和组织时必须慎重考虑, 以用于比较 EI 值, 特别是基因型之间。
未来应用
本文所描述的协议为研究人员提供了一个了解马铃薯 endoreduplication 的重要工具。它可能允许研究 endoreduplication 的遗传和环境成分, 在块茎组织中发展的时间过程, 以及对自然变化的评估。最终, endoreduplication 可能成为马铃薯改良的一个有希望的目标, 这项承诺将需要可靠的评估手段。

Endoreduplication 是细胞放弃典型细胞周期的过程, 取而代之的是经过反复重复 DNA 复制而没有细胞分裂的交替发展过程。由此产生的细胞将增加 DNA 含量和核大小, 这被认为在细胞调控、扩张和专业化中发挥作用。一般而言, 一轮 endoreduplication (称为 endocycle) 和相应增加的 dna 含量与较大的细胞体积相关, 观察, 沉淀的 "karyoplasmic 理论", 增加 DNA 含量需要适当调整更大的、可能更复杂的单元格1。这种现象在高等植物中很常见, 在一系列组织中被观察到, 包括结构/防御 (毛)2,3, 营养 (玉米胚乳)4,5和水槽/存储 (西红柿果皮;马铃薯块茎)6,7,8函数。在水果中, 有人建议 endoreduplication 在促进果皮的快速扩张方面起到了作用, endoreduplication 与果实发育期的负相关关系也证明了9。例如, DNA 含量高达512C 的细胞 (单倍体基因组的512倍) 已经在西红柿果皮8中观察到。此外, endoreduplication (2014) 表明, 细胞周期基因表达的改变可能导致果皮内的水平增加, 然后导致较大的水果10。因此, 改变基因促进 endoreduplication 提供了一个潜在的目标, 以改善生物量或产量通过植物育种或遗传操作。然而, 这种改进取决于对 endoreduplication 的原因和后果的进一步了解。
Endoreduplication 通常是通过流式细胞仪测量的, 即在粗组织制剂中释放的细胞核11, 用 DNA 结合荧光孵化, 如碘碘化12 (PI)。过滤后的样品然后通过激光的流式细胞仪, 其中的发射波长特定的荧光可以观察到。每个事件 (即,核) 的荧光强度与粒子的 DNA 含量直接相关。因此, 通过比较已知的标准, 可以计算给定样本中细胞的相对和绝对 DNA 含量。Endoreduplication 指数 (EI) 是通过观察细胞 dna 含量 (C 值) 来确定样本中每细胞 endocycles 的平均数量 (1C), 其中, 6.7 是单倍体细胞的 dna 含量 (该协议步骤中提出的公式)。例如, 在一个双倍的有机体中, 体细胞的基本 DNA 含量是2C。如果一个样本的细胞数为 4C, 相当于一轮 endoreduplication, 或更大, 它将有一个近0的 EI;然而, 如果几乎所有的细胞是4C 的 EI 将是大约1。然而, 由于多轮的 endoreduplication 是常见的高等植物观察到的 EI 值可能会更大。虽然计算 endoreduplication 指数可能比较简单, 但它要求可靠地确定核 C 值的相对丰度, 这在某些物种和组织 (包括马铃薯块茎) 中被排除。细胞解剖、化学或细胞壁组成的差异可能是13导致这些样品不符合典型的准备使用剃刀刀片将原子核直接释放到适当的缓冲, 通常使用与组织如西红柿果皮, 模型为 endoreduplication 研究8。
在马铃薯中, 对块茎内的 endoreduplication 的检查仍仅限于一对夫妇的研究, 也许部分原因是由于上述原油制剂产生不一致的结果而缺乏可靠的协议。尽管这种方法对树叶很管用, 但块茎样品经历了严重的退化和大量的碎片形式的噪音, 使得由不同 c-值的原子核组成的峰的分化几乎是不可能的。这可能是由于细胞组成的差异和块茎细胞内大量的细胞碎片 (e. g. 淀粉储存淀粉体)。为了克服这一障碍, 我们最近开发了一种更可靠的方法来获取马铃薯块茎流式细胞术中可分辨峰。每个峰值中的细胞频率可以用来计算不同基因型或不同组织组成块茎的准确 endoreduplication 水平。该协议采用了改进的原生质体提取方法14,15前面描述的流式细胞术11, 在马铃薯的亲属、品种和组织中显示出相当大的变化16.在这里, 我们通过评估在倍体, 组织和块茎大小的范围内的 EI 来详细介绍该协议。

<table>
	<tbody>
		<tr>
			<td>Serological pipette</td>
			<td>Fisher</td>
			<td>13-676-10k</td>
			<td>(sterile)</td>
		</tr>
		<tr>
			<td>Pipet Aid XP (autopipettor)</td>
			<td>Drummond Scientific</td>
			<td>4-000-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes (50 ml)</td>
			<td>Thermo Fisher Scientific</td>
			<td>339652</td>
			<td>(sterile)</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (1.5 ml)</td>
			<td>Globe Scientific</td>
			<td>111558</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (2 ml)</td>
			<td>Globe Scientific</td>
			<td>111552</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum filtration unit (0.22&#181;m)(aPES)</td>
			<td>Thermo Fisher Scientific</td>
			<td>564-0020</td>
			<td>(sterile)</td>
		</tr>
		<tr>
			<td>Vacuum filtration unit (0.45&#181;m)(aPES)</td>
			<td>Thermo Fisher Scientific</td>
			<td>168-0045</td>
			<td>(sterile)</td>
		</tr>
		<tr>
			<td>Cellulase &#34;Onozuka R-10&#34;</td>
			<td>Yakult</td>
			<td></td>
			<td>contact company</td>
		</tr>
		<tr>
			<td>Macerozyme &#34;Onozuka R-10&#34;</td>
			<td>Yakult</td>
			<td></td>
			<td>contact company</td>
		</tr>
		<tr>
			<td>Hemicellulase</td>
			<td>Sigma</td>
			<td>H2125</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MES buffer</td>
			<td>Acros Organics</td>
			<td>172590250</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>MP Biochemicals</td>
			<td>194854</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride (hexahydrate)</td>
			<td>Fisher Scientific</td>
			<td>M35-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (dihydrate)</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Acros Organics</td>
			<td>423922500</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Acros Organics</td>
			<td>17263-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate (monobasic, anhydrous)</td>
			<td>Sigma-Aldrich</td>
			<td>P-5379</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma-Aldrich</td>
			<td>P4170</td>
			<td>For flow cytometry preparations</td>
		</tr>
		<tr>
			<td>Ribonuclease A</td>
			<td>Sigma-Aldrich</td>
			<td>R5000</td>
			<td>For flow cytometry preparations</td>
		</tr>
		<tr>
			<td>Cork borers with punch</td>
			<td>American Scientific</td>
			<td>2093-02</td>
			<td>For smapling desired tissue</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Thermo Fisher Scientific</td>
			<td>16-31</td>
			<td>For smapling desired tissue</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Thermo Fisher Scientific</td>
			<td>08-920B</td>
			<td>For smapling desired tissue</td>
		</tr>
		<tr>
			<td>106um stainless steel mesh or other seive of same pore size</td>
			<td>New Jersey Wire Mesh Co.</td>
			<td></td>
			<td>contact company</td>
		</tr>
	</tbody>
</table>

measuring endoreduplication by flow cytometry of isolated tuber protoplasts

Endoreduplication, 复制细胞的核基因组没有随后的胞质分裂, 产生细胞的 DNA 含量增加, 并与专业化, 发展和细胞大小的增加有关。在植物中, endoreduplication 似乎促进了某些组织和器官的生长和扩展。其中包括马铃薯块茎 (马铃薯), 它在履行碳水化合物储存功能方面经历了相当大的细胞扩张。因此, endoreduplication 可以发挥重要作用, 块茎如何能够容纳这种丰富的碳。然而, 由于块茎的粗核分离方法所产生的细胞碎片, 可以有效地用于叶子的方法, 排除了对块茎 endoreduplication 指数 (EI) 的估计。本文提出了一种通过分离原生质体来评估块茎 endoreduplication 的技术, 同时证明了不同基因型和分离块茎组织的代表性结果。该协议的主要局限性是样品制备所需的时间和试剂成本, 以及在原生质体溶解后样品的寿命相对较短。虽然该议定书对技术变化很敏感, 但它代表了从这些大型专门细胞中分离出的传统核隔离方法的改进。提出了改进《议定书》的可能性, 如回收酶、使用护和其他改变。

原生质体生产
原生质体的产生是必要的, 以实现可重复流式细胞术的结果, 马铃薯块茎, 其一般形态学显示在图 1中。研究人员可能希望确保在添加 FCP 之前产生高质量的原生质体, 特别是在需要进行故障诊断的情况下。大多数原生质体应该是球状的, 最小的突起 (图 2), 并可能有很大的差异, 这也许是反映的差异在 EI。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57134/57134fig2.jpg"> 图 2: 在协议步骤4.4 中获得的具有代表性的叶和块茎原生质体.分离的原生质体应该是球形的, 与血浆膜保持对称。注意叶 (-c) 和块茎 (D, E) 原生质体之间的大小差异, 以及组织内的大小差异, 这可能表明不同级别的 endoreduplication。叶原生质体包含叶绿体, 而淀粉体在块茎原生质体内可见。刻度条 = 1000 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57134/57134fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
流式细胞术结果的评价
实验的成败可以从峰的宽度和它们在流式细胞术直方图中的分离来衡量。由于测量 endoreduplication 需要计算每个峰值中原子核的相对丰度, 如果有太多的噪声来绘制它们之间的有意义的边界, 那么仅仅存在或没有峰值就不够。图 3显示了研究者在流式细胞散射图和直方图中可能遇到的结果变化。在讨论中介绍了失败示例的潜在原因。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57134/57134fig3.jpg"> 图 3: 由完整和退化髓样的原生质体核流式细胞术获得的代表性结果.完整的原子核 (A) 显示峰值之间的干净分离, 以量化每个使用适当软件的单元格的相对丰度, 而退化样本 (B) 中的峰值显示移位、宽和重叠的基。在完整的 scatterplots (C) 和降级 (D) 示例中, 黑色框表示直方图的核事件门控, 事件的聚类反映在直方图峰值的宽度中。门外的事件表明碎片, 严重退化的细胞核, 或其他聚集物。PI H 对应于任意单位测量的荧光强度。在文本中讨论了防止样本退化的疑难解答方法。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57134/57134fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
Endoreduplication 组织间的差异
以前我们报告, 块茎髓组织有明显更大的 endoreduplication 水平比皮质组织16。为了确认和扩大这一点, 我们看了 endoreduplication 的三种不同的组织在 cv. 上级: 髓, perimedullary 实质, 和皮质, 复制三个, 每样2000门控事件。我们的结果证实了我们先前的观察, 髓组织有显著大于皮质组织的1.79 和 1.12 endocycles 每细胞的方法, 分别 (p = 0.018;学生的 t 测试)。有些令人惊讶的是, 软组织表现出类似于皮质的剖面, 也与髓组织有显著的不同 (平均 = 1.14; p = 0.013;学生的 t 测试)。这些结果在图 4中进行了总结。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57134/57134fig4.jpg"> 图 4: Endoreduplication 三个组织中的 cv 。叶的直方图 (A)、块茎皮质 (B)、薄壁 (C) 和髓 (D) 组织与从这些直方图计算的 EI 值一起显示。每个峰值的细胞核相对丰度的差异是显而易见的, 特别是在叶和块茎组织之间。同时显示每个峰值的 C 值。PI H 对应于任意单位测量的荧光强度。平均比较的块茎组织 EI 显示在 E 的星号表示显著差异 (p < 0.05) 和误差条显示标准偏差。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57134/57134fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
块茎大小和倍性的影响
我们以前报告说, 对于给定的基因型, 不同大小但相似成熟度的块茎并没有在 EI16中显示相应的差异。我们的目的是确认这一结果以及评估倍性和 endoreduplication 的关系。在这个实验中, 我们使用了三种不同基因型的三种软组织复制: cv (4x), VT_SUP_19 (2x), 这是从 cv 提取的双单倍体. 优于刺痛授粉 17, VT_SUP_19 4x, 这是一个加倍双单倍体18等基因系 VT_SUP_19。我们包括一组大 (90–130 g) 和小 (< 35 克) 块茎的复制为 cv. 优越的同时, 其他两种基因型的块茎是 90–130 g。这些块茎都是在完全成熟的温室植物中收获的,即植物的顶端有 senesced。我们观察到 VT_SUP_19 与其祖上级的显著差异 (p = 0.04);然而, VT_SUP_19 和 VT_SUP_19 4x 之间没有显著的差异 (p = 0.69)。这表明, 虽然有一个可能的遗传成分 endoreduplication, 如被揭露的基因组减少, 它不是由倍性, 至少在这种背景下。最后, 我们再次观察到大和小的 cv 之间没有显著的差异. 在图 5中演示的高级块茎。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57134/57134fig5.jpg"> 图 5: Endoreduplication 的两种大小的块茎, 其双 (VT_Sup_19) 和二、四倍体杂交 (VT_Sup_19 4x)衍生物。条形图显示的平均值为小 (< 35 克) 和大 (90–130 g) cv. 优越块茎并且大 (90–130 g) 块茎双单倍体 VT_SUP_19 和等基因系加倍双单倍体 VT_SUP_19 4x。重要区别 (p < 0.05) 由连接信件报告表明。误差线描述标准偏差。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57134/57134fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts at JoVE.com

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

分离块茎原生质体流式细胞仪测量 Endoreduplication 的研究

本文描述的协议是一种测量马铃薯块茎内 endoreduplication 的方法 (马铃薯)。它包括质壁分离和原生质体提取步骤, 以减少下游流细胞分析中的噪声和碎片。

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

measuring-endoreduplication-flow-cytometry-isolated-tuber

Nanyang Technological University

Research

JoVE Journal

Biology

10.1K 次观看.  Virginia Tech. 本文描述的协议是一种测量马铃薯块茎内 endoreduplication 的方法 (马铃薯)。它包括质壁分离和原生质体提取步骤, 以减少下游流细胞分析中的噪声和碎片。

视频: Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP - FCM:免疫沉淀，流式细胞仪检测

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

科研

生物学

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

代谢标记和流式细胞仪测定细胞周期进程的动力学

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

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

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

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

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

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

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

测量渗透水渗透系数（P<sub&gt; F</sub&gt;）球形细胞:隔离植物原生质体为例。

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

外层膜蛋白的分离线粒体的流式细胞仪免疫检测

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED热流量 - 结合光遗传学与流式细胞仪

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...