细胞周期进程的时间跟踪使用流式细胞仪，无需同步
Summary
这个协议描述了使用溴脱氧尿苷（BrdU）标记的摄取，以允许细胞是在S期在时间上的特定点的时间的跟踪。此外DNA染料和抗体标记的方便了S期细胞的命运在稍后时间详细分析。
Abstract
This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.
Introduction
的细胞周期功能和发生在细胞的变化过程中的细胞周期进展的评估是基本理解生物学，特别是癌症生物学的许多方面。在发展为恶性肿瘤的治疗许多药物对细胞周期进程产生深远的影响，或通过细胞周期依赖性机制诱导细胞死亡。为了研究细胞周期动力学或细胞在细胞周期的特定阶段中，通常以同步的细胞。然而同步方法可以对细胞有害作用被研究，有可能混淆的结果。1在单细胞随时间的细胞周期进程的最新的使用荧光标记的蛋白质，是只存在于细胞周期的特定阶段的已批准的分析2，但是所述细胞进行研究需要被遗传操纵以表达这些标记的蛋白质，限制了其使用的地方这可以读取系统容易完成。
细胞周期包括两个活性阶段：合成（S）相，其中，DNA复制和有丝分裂（M），其中，细胞分裂发生。这些阶段由三个间隙相，G 0，G 1和G 2分开。 ģ0或静止，是其中细胞已离开循环的休止阶段中，G 1是其中细胞大小之前的DNA复制和G 2，其中的DNA复制的完成之间但细胞分裂前的细胞生长继续增加。通过细胞周期的进展是由多个检查站的控制。当环境条件不支持DNA合成和防止进入S期对G 1检查点被激活。内-S期检查点或延迟可以通过DNA损伤可能会导致复制叉停滞了被触发。期间克2-复制的DNA的保真度被确认，并且如果检测到损坏那G 2 </子>检查点被激活允许细胞分裂DNA修复之前。有丝分裂期间的最后的检查点可确保染色单体已经在有丝分裂板被正确地对准，使得细胞分裂可顺利完成。这些检查点的3激活通常用于同步的细胞群。细胞周期关卡可以通过许多因素，但在肿瘤生物学中最常见的是检测DNA损伤激活。 DNA损伤响应由PI3激酶样激酶共济失调毛细血管扩张症和RAD3相关（ATR）发起和共济失调毛细血管扩张症突变（ATM），该激活下游效应激酶Chk1的和Chk2，分别3事件的范围激活的Chk1包括停滞复制叉，DNA的交联，和紫外线辐射的伤害而Chk2的是由双链断裂主要激活。
为研究对细胞周期i的长度的改变的条件下的效果的常用方法s到同步细胞在细胞周期的特定阶段。1这可以通过几种方法来实现。细胞可被物理分离基于尺寸，密度，侧向散射（粒度），以及细胞表面表达的标志。更实际，细胞可以通过化学手段进行同步。几剂，例如胸苷，羟基脲和阿糖胞苷可用于抑制DNA合成的细胞周期导致S期细胞继续循环的剂被除去后的累积的S期。细胞治疗诺考达唑，这防止形成有丝分裂纺锤体，逮捕与G 2 -或M相DNA含量。消除血清从培养基导致细胞于G1 0相位的累积。再加入内培养血清的营养素重新启动细胞的正常循环。然而，所有的这些同步方法干扰细胞的正常循环和生长，并且可以resul吨显著细胞死亡。
急性淋巴细胞白血病细胞的同步是特别具有挑战性的，并且这些细胞不适合于遗传操作。这里所描述的方法允许细胞周期动力学的评估和细胞在细胞周期的特定阶段没有传统同步或遗传修饰的研究。这种方法还可以用于其中，遗传修饰和传统的同步过程不容易实现其他类型的细胞是有用的。该方法是基于长期建立的使用溴脱氧尿苷（尿嘧啶）掺入，这对短期细胞的生长和增殖的影响非常小的。4成立的BrdU协议利用的BrdU的掺入在S期新合成的DNA 。这标志着永久细胞的BrdU曝光时已经处于S期。这部分人群可在稍后的时间点被染色的BrdU incorpor确定通货膨胀和由此充当可以遵循并评估在时间允许的对细胞周期转运药物作用的研究同步人口。 BrdU的需要前 抗体染色要曝光，通常是达到以下DNA酶或酸处理。6,7-使用流式细胞仪来检测掺入的BrdU使包括额外的标记。最重要的是利用染料来测量DNA含量，使该人在S期在研究开始时的细胞的细胞周期时相分布的评估。8另外附加的表面或细胞内的抗原，也可以研究。9，这些可能与细胞周期事件，如Ki67的或看似无关的细胞功能，如细胞凋亡的标记一样裂解的caspase-3。潜在的应用是由研究者的想象力的限制。
Protocol
Representative Results
Discussion
分析细胞周期的能力，对于癌症生物学的理解和两种药物和基因影响细胞增殖和生长的作用机制重要。虽然有许多测定法，据说测量细胞增殖的，多数只提供的量度指示的数目的细胞存在。这些包括测量通过直接可视化和计数，代谢活性或ATP浓度细胞数测定。许多这些方法的主要优点是，它们相对容易地进行，并适合于微孔板格式和自动化，使它们可用于筛选大量的条件或化合物。许多这些方法?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The work was funded by the Leukemia and Lymphoma Society of the USA (6105-08), a Cancer Council NSW grant (13-02), an NHMRC Senior Research Fellowship (LJB) (1042305) and project grant (1041614).
Materials
| APC BrdU Flow Kit | BD Biosciences | 552598 | Contains BrdU antibody, 7-AAD and BD Cytofix/Cytoperm Buffer (referred to as Fixation Buffer) |
| BD Cytoperm Permeabilization Buffer Plus | BD Biosciences | 561651 | Referred to as Permeabilization buffer |
| BD Perm/Wash Buffer | BD Biosciences | 554723 | Referred to as Wash buffer |
| DNase | Sigma | D-4513 | |
| BD Falcon 12 x 75-mm FACS tubes | BD Biosciences | 352008 | |
| BD Pharmingen Stain Buffer | BD Biosciences | 554656 | |
| BD LSR FORTESSA flow cytometer | BD Biosciences | FORTESSA | |
| Pipetman | Gilson | P2, P20, P100, P1000 | |
| RPMI 1640 w/o L-Gln 500 ml | Lonza | 12-167F | |
| DPBS | Lonza | 17-512F | |
| Fetal Bovine Serum | FisherBiotec | FBS-7100113 | |
| L-Glutamine | Sigma | G7513-100ML | |
| 5-Bromo-2′-deoxyuridine | Sigma | B5002-1G | |
| Falcon TC 150cm2 vented Flasks | BD Biosciences | 355001 | |
| Pipettes 25mL | Greiner | 760180 | |
| Aersol Pipettes 200µL | Interpath | 24700 | |
| Aersol Pipettes 1mL | Interpath | 24800 | |
| Centrifuge | Spintron | GT-175R | |
| CO2 incubator | Binder | C 150 | |
| AF488 anti-Histone H3 Phospho (Ser10) Antibody | Cell Signalling | 9708S | |
| Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb | Cell Signalling | 2197S | |
| Phospho-Chk1 (Ser345) (133D3) Rabbit mAb | Cell Signalling | 2348S | |
| NALM6 | DSMZ | ACC-128 |
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