Brdu细胞增殖检测试验

1、Brdu 检测细胞增殖实验实验操作 :1 . 铺细胞，每个 3.5cm dish 10 万个，在37、5%CO字箱中培养72h（细胞密度2 至 50-60%左右）。2 . Brdu （5-滨-2 '-脱氧尿音）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

2、：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 .力口一抗（尿口密噬脱氧核昔Brdu （鼠单抗）1:200 ）,用1%BSA&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:

3、100）,用 1%BS麻释，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PBSW释），避光室温反应10min。0 10min次，每次3洗PBS染好的细胞爬片用 DAPI.将15.16 .中性树胶封片，荧光显微镜观察，200X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DMS冲，储存浓度 为 100mg/ml 分装，

4、每管120ul ， -20 保存。b. 2 mol/L HCl :取 8.333mL 12 mol/L HCl 的浓 HCl,加入 DDWfe容至 50 mL。c. 0.1 mol/L 硼酸钠：称量 1.907g 硼砂（NaB- 10HO 381.36 g/mol ）,力口 入 DDW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 PBSW释

5、3%BSAS 1%g. 4%FPS： 4%多聚甲醛。我是用DDWE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使得一部分膜蛋白变

6、性， 从而使通透性进一步加强。 Triton-X-100 增加，并且利用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 区域，利用二抗连接不同的 证抗体识别的特异性 。二抗可以特异性识别一抗的 Fc 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DNA4、 M 个时期，其中S期是DN的成期，细胞内细月ft增殖周期包括G1、S、G2作为一种胸腺喀噬核昔BrdU进行半保留复制，各种构成DNA勺原料掺入到DNA中。原子连接的甲基被C 的类似物（

7、其化学结构特点是胸腺嘧啶的碱基嘧噬环上与5位合成中。当细胞处于 DNA臭代替），像胸腺口密噬核昔一样可掺入到细胞合成的DNA中，只要细胞掺入新合成的 DNABrdU在时，就会有BrdU期）而同时又有期（SBrdUBrdU可通过抗掺入到 DNA中长期存留。DNA勺不消亡，这种BrdU就在胞核的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DNAK链结构白位阻，BrdU抗体无法直接与双链上的BrdU结合，必须先用使 DNAB分变性，这本¥变性了的DNA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，

8、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是一种胸腺口密噬核昔类似物，能够在细胞增殖时期代替T渗入正在复制的DNA#子，通过基于 EdU与Apollo?荧光染料的特 异性反应检测DNAS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。的细胞只要不受 BrdU 该方法能对细胞周期进行迅速而稳定的测量, 而且标记到紫外线照射，对细胞本身没有功

9、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DNAM乱牛别是AT碱基结合，也可插入少于 3个连续AT碱基对的DNAf列中。当它与双链 DNA吉合时，荧光强度增强 20倍，而与单链DNA吉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 ；其特异性较漠化乙呢(ethidlium bromide , EB)和碘化丙噬

10、(propidium iodide , P1)高。DAPI的中文名称是4, 6-联眯-2-苯基口引噪，是一种常用 的荧光染料，其作用机理与漠化乙锭(EB)等染色剂的机理类似：它们与 DNAZ螺旋的凹槽部分可以发生相互作用，从而与DNA勺双链紧密结合。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 parameter

11、s toassess the cellular metabolism, physiology and pathology. Several techniques have been developed to quantitate these parameters utilizing thedifferential staining of fluorescent dyes.We are describing fourdifferentflow cytometric methods, two for the discrimination of cell cycle phasesapoptosisa

12、nd cycle cell of assessment simultaneous the for two and B) and (A(C and D).A) Bromodeoxyuridine/Propidium IodideThe classical method for the analysis of cell cycle distribution is theflow cytometric measurement of DNAcontent which can simultaneously determine the incorporation of Bromodeoxyuridine

13、(BrdU). The procedurerequires that DNAis partially denatured to expose incorporated BrdU to aspecific antibody. Denaturation is necessary because antibodies developedso far bind only to BrdU in single-strand DNA. The remaining undenaturedDNA is then stained with Propidium Iodide (PI). Green fluoresc

14、ence from thefluorescein-conjugated antibody is a measure of BrdU incorporation. Redfluorescence from the PI is a measure of DNA. The protocol described hereuses high-molarity HCl for the denaturation of DNA. Furthermore, this methodmay be utilizedeither for unfixed or for fixed cells in suspension.

15、B) Cyclins/Propidium IodideCyclins are key components of the cell cycle progression machinery. Inparticular, the expression of cyclins D, E, A and B1 provides newcell cyclelandmarks that can be used to subdivide cell cycle into severaldistinctsubcompartments. In this procedure cyclins expression is

16、detectable usingspecific monoclonal antibodies (mAbs), and is analysed in respect to DNAcontent.Generally, the peak of expression of cyclin D1 can be detected in earlyG1, the peak of cyclin E is typical of G1/S transition, the peakof cyclinA can be detected during G2/M phases and cyclin B1 is typica

17、l of late G2/M.Using this method, compared to the above mentioned protocol, it is possibleto distinguish G0 from G1 and G2 from M phases. However, it is necessaryto 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,

18、 evenif in avery few cell types).C) TUNEL/Propidium IodideOne of the most used protocol for the determination of apoptosis in thedifferent phases of cell cycle is the enzymatic in situ labelingof apoptosis-induced DNA strand breaks (TUNEL). Terminaldeoxynucleotidyltransferase (TdT) have been used fo

19、r the incorporation offluorescein-labeled nucleotides to DNAstrands breaks in situ . DNA contentis revealed by red fluorescence from PI. In order to have more details, seethe Chapters related to TUNEL technique.D) F-Actin/Propidium IodideThe analysis of apoptotic cells and estimation of their cell c

20、yclespecificity is also possible using a recent method. This is based onidentification of apoptotic cells which have modified theircytoskleton andtheir DNA content. In specific, paraformaldehyde (PFA) fixation followedby staining of F-actin with fluorescein-conjugated phalloidin andof DNAwith PI, ar

21、e 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. Resu

22、spend 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 ofmAbant-BrdU.9. Incubate for 1 hour at

23、4° C in the dark.10. As in step 5.11. Resuspend in 200 m L of washing buffer and label with 4 m Lofgoat-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 mL of washing buffer and 200 mL of PI buffer.C in the dark.15. Incub

24、ate for 15-30 min at 416. Analyse with flow cytometer equipped with a 488 nmargon laser.A.3. COMMENTARYA.3.1 Background informationIn this procedure fixed cells by 4%PFAin Phosphate Buffer Saline(PBS)can be utilized. In this case to wash cells once in PBS before tostart atstep 1 is necessary.Moreover, both direct and indirect immunofluorescence can be