2014-5-6 PI法细胞周期检测

2014-5-6 PI法细胞周期检测 细胞周期检测(PI法) 一、实验方法原理 细胞周期(cell cycle):是指细胞从前一次分裂结束起到下一次分裂结束为止的活动过程，通常由G0/G1期、S期、G2期和M期组成。G1期:细胞开始RNA和蛋白质的合成，但DNA含量仍保持二倍体。S期:DNA开始合成，这时细胞核内DNA的含量介于G1期和G2期之间。当DNA复制结束成为4倍体时，细胞进入G2期。G2期的细胞继续合成RNA及蛋白质，直到进入M期。因此，单纯从DNA含量无法区分G2期和M期。一旦有丝分裂发生，细胞分裂成两个细胞，这两个细胞或者进入下一个细胞周期，或者进入静止期(G0期)，而G0期从DNA含量上同样无法与G1期区分。因此，整个复制周期可以描述为G0/G1、S、G2/M期。通过核酸染料PI标记DNA,并由流式细胞仪进行 分析 定性数据统计分析pdf销售业绩分析模板建筑结构震害分析销售进度分析表京东商城竞争战略分析 ，可以得到细胞各个时期的分布状态，计算出G0/G1%、S%、G2/M%，了解增殖能力，在肿瘤病理学研究中，通常以S期细胞比率作为判断肿瘤增殖状态的指标。 流式细胞仪的工作原理:是将待测细胞放入样品管中，在气体的压力下进入充满鞘液的流动室。在鞘液的约束下细胞排成单列由流动室的喷嘴喷出，形成细胞柱。通过对流动液体中排列成单列的细胞进行逐个检测，得到该细胞的光散射和荧光指标，分析出其DNA特征。 细胞周期检测的原理:PI法是较常见的一种周期检测方法。PI为插入性核酸荧光染料，能选择性的嵌入核酸DNA和RNA双链螺旋的碱基之间与之结合，其结合的量与DNA的含量成正比例关系，用流式细胞仪进行分析，就可以得到细胞周期各个阶段的DNA分布状态，从而计算出各个期的百分含量。PI染色后，假设G0/G1期细胞的荧光adership: Luo Mingjun, rectification time: before September 25th, insist for a long time) 1, 2, to further improve research, solve thoroughly, not solid, the effect is not obvious. The major issues and enterprises it is a matter of security bureau year development, the vital interests of the problem, determine the research topic, by members of the Party led research. Basic research to see the truth, to the truth, do not say hello, not pre arranged. After the investigation to submit the research report. 3, insist on grassroots party members take the lead in implementing the line. <. In the construction of work style building long-term mechanism views > and < on the depth of the grassroots, the people, promoting harmony activities notice >, more mines and enterprises, solve the masses of grassroots leaders, mine is not enough. 4, contact a crowd. Combined with the "grassroots cadres contact farmers" activities and each cadres to contact a people, by 强度为1，那么含有双份基因组DNA的G2/M期细胞的荧光强度的理论值为2，正在进行DNA复制的S期细胞的荧光强度为1-2之间。 细胞周期示意图如(图一)所示 (图一) 二、 材料 关于××同志的政审材料调查表环保先进个人材料国家普通话测试材料农民专业合作社注销四查四问剖析材料 及准备工作 1.材料准备 试剂、耗材名称 品牌 货号 细胞培养瓶 corning 6孔板 corning 10ml离心管 国产 1.5ml EP管 国产 胰酶(无EDTA) 贝博试剂盒中包括 RNase-A 贝博试剂盒中包括 PI 贝博试剂盒中包括 无水乙醇 国产 PBS 自配 adership: Luo Mingjun, rectification time: before September 25th, insist for a long time) 1, 2, to further improve research, solve thoroughly, not solid, the effect is not obvious. The major issues and enterprises it is a matter of security bureau year development, the vital interests of the problem, determine the research topic, by members of the Party led research. Basic research to see the truth, to the truth, do not say hello, not pre arranged. After the investigation to submit the research report. 3, insist on grassroots party members take the lead in implementing the line. <. In the construction of work style building long-term mechanism views > and < on the depth of the grassroots, the people, promoting harmony activities notice >, more mines and enterprises, solve the masses of grassroots leaders, mine is not enough. 4, contact a crowd. Combined with the "grassroots cadres contact farmers" activities and each cadres to contact a people, by 2. 试剂配制 10XPBS液体配方(0.1MPBS pH 7.4) 以1L量为例: NaCl 80g Na2HPO4?12H2O 32.3g NaH2PO4?2H2O 4.5g (十二水合磷酸氢二钠和二水合磷酸二氢钠) 加双蒸水900mL 左右，用HCl/NaOH 调pH至7.4，最后定容为1000mL。 使用时用去离子水稀释成1XPBS 3.仪器设备 流式细胞仪;细胞培养孵箱;离心机;水浴;冰箱;离心管;封口膜;移液器 三、 实验步骤及注意事项:(采用贝博公司的细胞周期检测试剂盒) 1. 细胞样品的准备: 6? 单次流式细胞仪检测，1份样品细胞数量约10(我校8楼免疫教 研室的要求) ? 收集细胞培养液备用。 ? 用胰酶(无EDTA)消化细胞，至细胞可以被轻轻用吹打下来时， 加入前面收集的细胞培养液，吹打下所有的贴壁细胞，并轻轻吹 散细胞。 ? 将细胞悬液收集到10ml的离心管内。800g左右离心5min，沉淀 细胞。小心吸除上清，残留约50微升左右的培养液，以避免吸走 细胞。 ? 加入1ml冰浴预冷的PBS，重悬细胞， 800g×5min。为减少细胞 损失可用1.5ml离心。 adership: Luo Mingjun, rectification time: before September 25th, insist for a long time) 1, 2, to further improve research, solve thoroughly, not solid, the effect is not obvious. The major issues and enterprises it is a matter of security bureau year development, the vital interests of the problem, determine the research topic, by members of the Party led research. Basic research to see the truth, to the truth, do not say hello, not pre arranged. After the investigation to submit the research report. 3, insist on grassroots party members take the lead in implementing the line. <. In the construction of work style building long-term mechanism views > and < on the depth of the grassroots, the people, promoting harmony activities notice >, more mines and enterprises, solve the masses of grassroots leaders, mine is not enough. 4, contact a crowd. Combined with the "grassroots cadres contact farmers" activities and each cadres to contact a people, by ? 再次离心沉淀细胞，小心吸除上清，残留约20ul左右的PBS，以 避免吸走细胞。重复第5步操作，用预冷的PBS再次洗细胞，并离心收集细胞。轻轻弹击离心管底以适当分散细胞，避免细胞成团，用250ul预冷的PBS重悬细胞。 2. 细胞固定: 缓慢加入750ul冰浴预冷无水乙醇，轻轻吹打混匀，4?固定 2小时或-20?固定1小时，封口膜封口(此步可停止，-20?保存 样品，1周内样品检测不受影响)。 3. 染色: ?1000g左右离心3-5min，沉淀细胞。小心吸除上清，可以残留约50ul左右的70%乙醇，以避免吸走细胞。 ?加入约1ml冰浴预冷的PBS，洗涤细胞一次。再次离心沉淀细胞，小心吸除上清。 ?用200ul预冷的PBS重悬细 ?加入Rnase A溶液20ul，37?水浴30min。 ? 每管细胞样品中加入400ul碘化丙啶染色液，缓慢并充分混匀后4?避光孵育30 min。染色完成后宜在24h内完成流式检测。 ?. 流式检测和分析:用流式细胞仪在激发波长488nm波长处检测红色荧光，同时检测光散射情况。采用适当分析软件进行细胞DNA含量分析和光散射分析。 注意事项 1.RNaseA用PBS配制，但是注意要沸水浴去除DNase的活性。试剂盒中带有配制好的酶。 2.流式分析时需要的是单个细胞悬液，因此在操作过程中充分混悬细 adership: Luo Mingjun, rectification time: before September 25th, insist for a long time) 1, 2, to further improve research, solve thoroughly, not solid, the effect is not obvious. The major issues and enterprises it is a matter of security bureau year development, the vital interests of the problem, determine the research topic, by members of the Party led research. Basic research to see the truth, to the truth, do not say hello, not pre arranged. After the investigation to submit the research report. 3, insist on grassroots party members take the lead in implementing the line. <. In the construction of work style building long-term mechanism views > and < on the depth of the grassroots, the people, promoting harmony activities notice >, more mines and enterprises, solve the masses of grassroots leaders, mine is not enough. 4, contact a crowd. Combined with the "grassroots cadres contact farmers" activities and each cadres to contact a people, by 胞。 3.固定过程中不直接加70%乙醇的原因是:直接加入乙醇会导致细胞团聚的现象，很难重悬成单细胞。乙醇固定之后没有细胞沉淀。 3. PI液体4?避光保存。 FlowJo分析细胞周期数据的过程 1.把细胞周期样品拖入FlowJo软件，双击打开原始数据。如(图二) 所示: (图二) X轴选择FSC,Y轴选择SSC。分析细胞周期的细胞选择上图所示的细胞群体进行分析。 2. 在SSC/FSC图中双击分析的细胞群体，如(图三)所示: adership: Luo Mingjun, rectification time: before September 25th, insist for a long time) 1, 2, to further improve research, solve thoroughly, not solid, the effect is not obvious. The major issues and enterprises it is a matter of security bureau year development, the vital interests of the problem, determine the research topic, by members of the Party led research. Basic research to see the truth, to the truth, do not say hello, not pre arranged. After the investigation to submit the research report. 3, insist on grassroots party members take the lead in implementing the line. <. In the construction of work style building long-term mechanism views > and < on the depth of the grassroots, the people, promoting harmony activities notice >, more mines and enterprises, solve the masses of grassroots leaders, mine is not enough. 4, contact a crowd. Combined with the "grassroots cadres contact farmers" activities and each cadres to contact a people, by (图三) X轴选择FL3-LIN,用来表示PI的线性信号。Y轴选择FL3-PEAK,用来表示PI的峰值信号。本实验数据采集采用的是贝克曼仪器采集的数据。(如果是BD的仪器采集的数据，X轴选择FL2-W,用来表示PI的宽度信号。Y轴选择FL2-A,用来表示PI的面积信号)，如上图所示多倍体或异倍体细胞不予分析。 3.在FlowJo工作台中选中“用于周期分析的二倍体细胞”这个结点，在工具栏的“工具”中选择“细胞周期”。 如(图四)所示 (图四) 说明: 1) 分析的模型有Watson和DJF两种，用户可以选择任意一种进行 分析，两模型间没有实质的差别。模式右边是两种模型的显示方 式。 2) 细胞周期拟合原则:G1峰限制和G2峰限制。根据实验结果拟合 情况，对G1峰限制和G2峰限制进行适当的调整。 adership: Luo Mingjun, rectification time: before September 25th, insist for a long time) 1, 2, to further improve research, solve thoroughly, not solid, the effect is not obvious. The major issues and enterprises it is a matter of security bureau year development, the vital interests of the problem, determine the research topic, by members of the Party led research. Basic research to see the truth, to the truth, do not say hello, not pre arranged. After the investigation to submit the research report. 3, insist on grassroots party members take the lead in implementing the line. <. In the construction of work style building long-term mechanism views > and < on the depth of the grassroots, the people, promoting harmony activities notice >, more mines and enterprises, solve the masses of grassroots leaders, mine is not enough. 4, contact a crowd. Combined with the "grassroots cadres contact farmers" activities and each cadres to contact a people, by 曲线调整原则:粉色的曲线为模型曲线，黑色的为样本曲线，理想状况是粉色和黑色曲线吻合一致为最佳。拟和的好坏可以 , 根据右边的RMS值来判断。RMS(root mean square)值越小 越好。但是这只是一个相对值，只在同样本不同模拟情况下， 选取最小值来达到最佳拟和。你可以选中G1期的峰或G2 期的峰，根据调节按钮调节峰的宽度 , 通常来说，G2的位置为G1的2倍。 你可以根据相对固定 的那个峰来确定另外一个峰的位置，如G1峰相对固定的话， 就选择G2峰的均数为G1的2倍(2G1);如果G2峰相对固定 的话，就选择G1峰为G2峰的0.5G2细胞周期结果解读: G0/G1期细胞占总的61.2%，峰位于横座标的45.76 G2/M期占13.07，峰位于横座标的91.43 S期占25.73 G2/G1为2.0(即G2期为4倍体细胞，而G1期为2倍体细胞，比值为2) 峰的变异系数为4.54%(好) 细胞碎片为0.48%，细胞聚集体有0.06%。 总的细胞数(仪器检测到的)为17525个， adership: Luo Mingjun, rectification time: before September 25th, insist for a long time) 1, 2, to further improve research, solve thoroughly, not solid, the effect is not obvious. The major issues and enterprises it is a matter of security bureau year development, the vital interests of the problem, determine the research topic, by members of the Party led research. Basic research to see the truth, to the truth, do not say hello, not pre arranged. After the investigation to submit the research report. 3, insist on grassroots party members take the lead in implementing the line. <. In the construction of work style building long-term mechanism views > and < on the depth of the grassroots, the people, promoting harmony activities notice >, more mines and enterprises, solve the masses of grassroots leaders, mine is not enough. 4, contact a crowd. Combined with the "grassroots cadres contact farmers" activities and each cadres to contact a people, by 在细胞周期中分析的细胞数为17431个(即排除了碎片及聚集体后) CV是变异系数。一般CV越小，峰形越好，越尖锐。能控制在5%左右是比较好的结果，一般小于10%就可以认可了。 细胞周期分析常用术语: , CV:变异系数。不同细胞群体DNA含量的细微差别可能有生物 学上的重要意义，因此DNA含量的流式分析中分辨率尤其关 键。通常检测分辨率或精度由G0/G1峰的CV来反映。影响CV 的因素主要包括两方面:仪器因素和样品制备及染色过程对标 本的人为影响。 , Ploidy:倍体。原指染色体数目。流式中用来描述总的DNA含量。 二倍体细胞有正常细胞的DNA 含量但不排除染色体的异常。 S期含量 :S期细胞占总细胞周期的比例。 细胞周期数据结果输出 1. 细胞周期结果的输出根据最终的需求可以有多种输出方式 , 在细胞周期分析窗口工具栏中选择“文件”并单击，选择下拉 菜单中的“另存为”，选择保存位置、重新命名文件名以及文 件保存的文件类型(SVG、PNG、EMF、JPG)。 把上述样品的分析应用到组中，在Flow Jo软件界面中单击打开布局编辑器，在工作台的样品空间把“分析的细胞群体结点”拖入布局编辑器中，单击布局编辑器右上角的Batch工具。输出整个实验组的数据。如(图五)所示 adership: Luo Mingjun, rectification time: before September 25th, insist for a long time) 1, 2, to further improve research, solve thoroughly, not solid, the effect is not obvious. The major issues and enterprises it is a matter of security bureau year development, the vital interests of the problem, determine the research topic, by members of the Party led research. Basic research to see the truth, to the truth, do not say hello, not pre arranged. After the investigation to submit the research report. 3, insist on grassroots party members take the lead in implementing the line. <. In the construction of work style building long-term mechanism views > and < on the depth of the grassroots, the people, promoting harmony activities notice >, more mines and enterprises, solve the masses of grassroots leaders, mine is not enough. 4, contact a crowd. Combined with the "grassroots cadres contact farmers" activities and each cadres to contact a people, by (图五) adership: Luo Mingjun, rectification time: before September 25th, insist for a long time) 1, 2, to further improve research, solve thoroughly, not solid, the effect is not obvious. The major issues and enterprises it is a matter of security bureau year development, the vital interests of the problem, determine the research topic, by members of the Party led research. Basic research to see the truth, to the truth, do not say hello, not pre arranged. After the investigation to submit the research report. 3, insist on grassroots party members take the lead in implementing the line. <. In the construction of work style building long-term mechanism views > and < on the depth of the grassroots, the people, promoting harmony activities notice >, more mines and enterprises, solve the masses of grassroots leaders, mine is not enough. 4, contact a crowd. Combined with the "grassroots cadres contact farmers" activities and each cadres to contact a people, by