Brdu细胞增殖检测实验

Brdu检测细胞增殖实验实验操作:孵箱中培养72h（细胞密1．铺细胞，每个3.5cm dish 10万个，在37℃、5%CO2度至50-60%左右）。

2．Brdu（5-溴-2′-脱氧尿苷）加入培养细胞中，1mg/ml，标记48h。

(量：Brdu 以铺满整个dish底面为准。

)3．固定：PBS洗细胞爬片3次，每次5min，在摇床上晃动清洗，4%PFA固定30min。

4．变性：将固定好的细胞爬片用PBS洗3次，每次5min，2mol/L的HCl在37℃条件下变性5min，可放置于37℃恒温孵箱，应用封口膜把培养皿封好。

（120r/m）5．中和：0.1mol/L的硼酸钠（PH8.3）中和10min，PBS洗3次，每次5min。

（50r/m）6．加入1ml的0.2%TritonX-100，10min。

7．吸出TritonX-100，用PBS洗3次，每次5min。

8．加入1ml 3%的BSA封闭，室温1h，可在摇床上晃动。

9．吸出BSA，用PBS洗3次，每次5min。

10．加一抗（尿嘧啶脱氧核苷Brdu（鼠单抗）1:200），用1%BSA稀释，4度过夜。

11．将孵好一抗的细胞爬片用PBS洗3次，每次10min。

12．加二抗（羊抗鼠IgG/Alexa Fluor 594 1: 100），用1%BSA稀释，避光室温孵育1h。

（60 r/min）13．将孵好二抗的细胞爬片用PBS洗3次，每次10min。

14．加DAPI染细胞核，储存浓度为1mg/ml，应将DAPI完全混匀，可用手弹几下，一般稀释比例为1:1000（用PBS稀释），避光室温反应10min。

15．将DAPI染好的细胞爬片用PBS洗3次，每次10min。

16．中性树胶封片，荧光显微镜观察，200×镜下取5个视野，计数Brdu阳性细胞和蓝染的细胞核数目，然后进行统计分析。

试剂配制：a. Brdu 的溶解：室温下，将250mg 粉末溶于2.5ml 的DMSO 中，储存浓度为100mg/ml分装，每管120ul ，-20℃保存。

b. 2 mol/L HCl ：取8.333mL 12 mol/L HCl 的浓HCl ，加入DDW 定容至50 mL 。

c. 0.1 mol/L 硼酸钠：称量1.907g 硼砂（Na 2B 4·10H 2O 381.36 g/mol ），加入DDW定容至50 mL ，调PH=8.3。

d. 0.2% Triton X-100：有0.5%的 Triton-100（2.5 mL 原液溶解于47.5 mL 的PBS 中），用PBS 稀释至0.2%。

e. 3% BSA ：称量1.5g BSA ，溶解于50 mL 的PBS 中。

f. 1% BSA ：用PBS 稀释3%BSA 至1%。

g. 4%FPS ：4%多聚甲醛。

我是用DDW 配制的，后来我发现很难溶，磁力搅拌器加热搅拌，虽然温度控制在60℃以下，也总是担心多聚甲醛分解为甲醛，所以，我就总结为如下：提前配制。

4%多聚甲醛溶液（pH7.2） 试剂：多聚甲醛（PFA ） 4g DDW 至100ml 配制方法：称取4g 多聚甲醛（粉末状），置于三角烧瓶中，加入80mlDDW ，放入37恒温水浴箱，每隔1-2小时摇晃混匀，16-24小时PFA 会完全溶解。

补充DDW ，调节PH 值。

实验原理：1. 免疫染色实验的基本原理利用固定剂（通常是甲醛或多聚甲醛）将细胞固定，使得细胞膜的通透性大大增加，并且利用Triton-X-100使得一部分膜蛋白变性，从而使通透性进一步加强。

利用正常羊血清封闭，可以令许多蛋白先与血清内的非特异性抗体结合，而特异性的抗体由于动力学的关系可以通过竞争性的反应与目的蛋白结合，这一过程可以保证抗体识别的特异性。

二抗可以特异性识别一抗的Fc区域，利用二抗连接不同的荧光基团，就可以在荧光显微镜下观察到不同的荧光，从而显示目的基因的表达情况。

2.BrdU标记原理细胞增殖周期包括G1、S、G2、M 4个时期，其中S期是DNA合成期，细胞内DNA进行半保留复制，各种构成DNA的原料掺入到DNA中。

BrdU作为一种胸腺嘧啶核苷的类似物（其化学结构特点是胸腺嘧啶的碱基嘧啶环上与5位C原子连接的甲基被溴代替），像胸腺嘧啶核苷一样可掺入到细胞合成的DNA中。

当细胞处于DNA合成期（S期）而同时又有BrdU存在时, 就会有BrdU掺入新合成的DNA中，只要细胞不消亡，这种BrdU就在胞核的DNA中长期存留。

掺入到DNA的BrdU可通过抗BrdU单克隆抗体在组织切片或细胞爬片上显示。

BrdU抗体比较大，由于DNA双链结构的位阻，BrdU抗体无法直接与双链上的BrdU结合，必须先用使DNA部分变性，这样变性了的DNA单链上的BrdU才能与BrdU抗体结合，因此做BrdU细胞增殖实验一定要变性，当然变性的方法包括酸解，热解等，但是要注意变性的程度也很重要。

建议采用EdU细胞增殖检测方法，无需变性，无需酶解，无需抗体，小分子染色，3小时完成实验。

EdU是一种胸腺嘧啶核苷类似物，能够在细胞增殖时期代替T渗入正在复制的DNA分子，通过基于EdU与Apollo?荧光染料的特异性反应检测DNA复制活性，通快速、更灵敏、更准确。

EdU检测染料只有BrdU抗体大小的1/500，在细胞内很容易扩散，无需DNA 变性（酸解、热解、酶解等）即可有效检测，可有效避免样品损伤，在细胞和组织水平能更准确地反映细胞增殖等现象。

该方法能对细胞周期进行迅速而稳定的测量, 而且标记BrdU的细胞只要不受到紫外线照射，对细胞本身没有功能损害。

该技术可应用到跟踪检测移植细胞的存活、分化和功能状态。

3.DAPI染色原理DAPI为4’，6二脒基-2-苯吲哚(4’，6—diamidino-2—phenylindole)，能与双链DNA小槽，特别是AT碱基结合，也可插入少于3个连续AT碱基对的DNA 序列中。

当它与双链DNA结合时，荧光强度增强20倍，而与单链DNA结合则无荧光增强现象，因此是一种简易、快速和敏感地检测DNA的方法。

DAPI的荧光强度虽较Hoechst低，但荧光稳定性优于Hoechst；其特异性较溴化乙啶(ethidlium bromide，EB)和碘化丙啶(propidium iodide，P1)高。

DAPI的中文名称是4，6-联脒-2-苯基吲哚，是一种常用的荧光染料，其作用机理与溴化乙锭（EB）等染色剂的机理类似：它们与DNA双螺旋的凹槽部分可以发生相互作用，从而与DNA的双链紧密结合。

结合后产生的荧光基团的吸收峰是358nm而散射峰是461nm，正好UV（紫外光）的激发波长是356nm，使得DAPI成为了一种常用的荧光检测信号。

ANALYSIS OF CELL CYCLE1. INTRODUCTIONCell cycle and apoptosis are very important functional parameters to assess the cellular metabolism, physiology and pathology. Several techniques have been developed to quantitate these parameters utilizing the differential staining of fluorescent dyes. We are describing four different flow cytometric methods, two for the discrimination of cell cycle phases (A and B) and two for the simultaneous assessment of cell cycle and apoptosis (C and D).A) Bromodeoxyuridine/Propidium IodideThe classical method for the analysis of cell cycle distribution is the flow cytometric measurement of DNA content which can simultaneously determine the incorporation of Bromodeoxyuridine (BrdU). The procedure requires that DNA is partially denatured to expose incorporated BrdU to a specific antibody. Denaturation is necessary because antibodies developed so far bind only to BrdU in single-strand DNA. The remaining undenatured DNA is then stained with Propidium Iodide (PI). Green fluorescence from the fluorescein-conjugated antibody is a measure of BrdU incorporation. Red fluorescence from the PI is a measure of DNA. The protocol described here uses high-molarity HCl for the denaturation of DNA. Furthermore, this method may be utilized either for unfixed or for fixed cells in suspension.B) Cyclins/Propidium IodideCyclins are key components of the cell cycle progression machinery. In particular, the expression of cyclins D, E, A and B1 provides new cell cycle landmarks that can be used to subdivide cell cycle into several distinct subcompartments. In this procedure cyclins expression is detectable using specific monoclonal antibodies (mAbs), and is analysed in respect to DNA content.Generally, the peak of expression of cyclin D1 can be detected in early G1, the peak of cyclin E is typical of G1/S transition, the peak of cyclinA can be detected during G2/M phases and cyclin B1 is typical of late G2/M. Using this method, compared to the above mentioned protocol, it is possible to distinguish G0 from G1 and G2 from M phases. However, it is necessary to keep in mind that not all cell types behave in the same manner (for example, cyclin D1 is detectable not only in G0/G1 but also in G2/M, even if in a very few cell types).C) TUNEL/Propidium IodideOne of the most used protocol for the determination of apoptosis in the different phases of cell cycle is the enzymatic in situ labeling of apoptosis-induced DNA strand breaks (TUNEL). Terminal deoxynucleotidyl transferase (TdT) have been used for the incorporation of fluorescein-labeled nucleotides to DNA strands breaks in situ . DNA content is revealed by red fluorescence from PI. In order to have more details, see the Chapters related to TUNEL technique.D) F-Actin/Propidium IodideThe analysis of apoptotic cells and estimation of their cell cycle specificity is also possible using a recent method. This is based on identification of apoptotic cells which have modified their cytoskleton and their DNA content. In specific, paraformaldehyde (PFA) fixation followed by staining of F-actin with fluorescein-conjugated phalloidin and of DNA with PI, are used. Furthermore, this procedure may be utilized also for adherent cells.A) BrdU/PI PROTOCOLA.2.1 Materials1. Cells (1x106 /mL) are incubated with BrdU 10 m M at final concentration, for 30 min at 37 °C in controlled atmosphere.2.Wash twice at 500 g for 1 min using the washing buffer.3. Resuspend in 0.5 mL of washing buffer and 0.5 mL of HCl 4 M.4. Mix accurately and incubate for 30 min at room temperature.5. Wash once as in step 2.6. Resuspend in 1 mL of Borax buffer.7. As in step 5.8. Resuspend in 200 m L of washing buffer and label with 5 m L of mAb ant-BrdU.9. Incubate for 1 hour at 4 °C in the dark.10. As in step 5.11. Resuspend in 200 m L of washing buffer and label with 4 m L of goat-anti-mouse FITC-conjugated antibody.12. Incubate for 30 min at 4 °C in the dark.13. As in step 5.14. Resuspend in 200 m L of washing buffer and 200 m L of PI buffer.15. Incubate for 15-30 min at 4 °C in the dark.16. Analyse with flow cytometer equipped with a 488 nm argon laser.A.3. COMMENTARYA.3.1 Background informationIn this procedure fixed cells by 4% PFA in Phosphate Buffer Saline (PBS) can be utilized. In this case to wash cells once in PBS before to start at step 1 is necessary.Moreover, both direct and indirect immunofluorescence can be used. The BrdU incorporation is more evident using the indirect method.A.3.2 Anticipated resultsIt is recommanded to perform each experiment using a negative and a positive control sample. The negative sample, in order to have a correct setting of instrument, is assessed following all the steps except step 8. The positive sample, in order to make sure that the method works, is assessed by using a proliferating cell line (such as U937, K562, MOLT4, etc.) following all steps. A.3.3 Time considerationsThe protocol is simply but it require a quite long time. Indeed, for few samples, more or less 4 hours are required. The duration of the method is obviously depending on the number of samples. A.3.4 Key references1. Dolbeare, F., Gratzner, H:, Pallavicini, M., Gray, J.W. 1983. Proc. Natl. Accad. Sci. U.S.A .80: 5573.。