細胞增殖檢測實驗

1、Brdu檢測細胞增殖實驗實驗操作:1 鋪細胞，每個3.5cm dish 10萬個，在37、5%CO2孵箱中培養(yǎng)72h（細胞密度至50-60%左右）。2 Brdu（5-溴-2-脫氧尿苷）加入培養(yǎng)細胞中，1mg/ml，標記48h。(量：Brdu以鋪滿整個dish底面為準。)3 固定：PBS洗細胞爬片3次，每次5min，在搖床上晃動清洗，4%PFA固定30min。4 變性：將固定好的細胞爬片用PBS洗3次，每次5min，2mol/L的HCl在37條件下變性5min，可放置于37恒溫孵箱，應(yīng)用封口膜把培養(yǎng)皿封好。（120r/m）5 中和：0.1mol/L的硼酸鈉（PH8.3）中和10min，PBS洗3

2、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封閉，室溫1h，可在搖床上晃動。9 吸出BSA，用PBS洗3次，每次5min。10加一抗（尿嘧啶脫氧核苷Brdu（鼠單抗）1:200），用1%BSA稀釋，4度過夜。11將孵好一抗的細胞爬片用PBS洗3次，每次10min。12加二抗（羊抗鼠IgG/Alexa Fluor 594 1: 100），用1%BSA稀釋，避光室溫孵育1h。（60 r/min）13將孵好二抗的細胞爬片用PBS洗3次，每次10min。14加DAP

3、I染細胞核，儲存濃度為1mg/ml，應(yīng)將DAPI完全混勻，可用手彈幾下，一般稀釋比例為1:1000（用PBS稀釋），避光室溫反應(yīng)10min。15將DAPI染好的細胞爬片用PBS洗3次，每次10min。16中性樹膠封片，熒光顯微鏡觀察，200×鏡下取5個視野，計數(shù)Brdu陽性細胞和藍染的細胞核數(shù)目，然后進行統(tǒng)計分析。試劑配制：a. Brdu的溶解：室溫下，將250mg粉末溶于2.5ml的DMSO中，儲存濃度為100mg/ml分裝，每管120ul，-20保存。b. 2 mol/L HCl：取8.333mL 12 mol/L HCl的濃HCl，加入DDW定容至50 mL。c. 0.1 mo

4、l/L硼酸鈉：稱量1.907g硼砂（Na2B4·10H2O 381.36 g/mol），加入DDW定容至50 mL，調(diào)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稀釋至0.2%。e. 3% BSA：稱量1.5g BSA，溶解于50 mL的PBS中。f. 1% BSA：用PBS稀釋3%BSA至1%。g. 4%FPS：4%多聚甲醛。我是用DDW配制的，后來我發(fā)現(xiàn)很難溶，磁力攪拌器加熱攪拌，雖然溫度控制在60以下，也總是擔心多聚甲醛分解為甲醛，所以，我就總結(jié)為如下：提前配制。4%多聚甲

5、醛溶液（pH7.2） 試劑：多聚甲醛（PFA） 4g DDW 至100ml配制方法：稱取4g多聚甲醛（粉末狀），置于三角燒瓶中，加入80mlDDW，放入37恒溫水浴箱，每隔1-2小時搖晃混勻，16-24小時PFA會完全溶解。補充DDW，調(diào)節(jié)PH值。實驗原理：1. 免疫染色實驗的基本原理利用固定劑（通常是甲醛或多聚甲醛）將細胞固定，使得細胞膜的通透性大大增加，并且利用Triton-X-100使得一部分膜蛋白變性，從而使通透性進一步加強。利用正常羊血清封閉，可以令許多蛋白先與血清內(nèi)的非特異性抗體結(jié)合，而特異性的抗體由于動力學(xué)的關(guān)系可以通過競爭性的反應(yīng)與目的蛋白結(jié)合，這一過程可以保證抗體識別的特異性

6、。二抗可以特異性識別一抗的Fc區(qū)域，利用二抗連接不同的熒光基團，就可以在熒光顯微鏡下觀察到不同的熒光，從而顯示目的基因的表達情況。2. BrdU標記原理細胞增殖周期包括G1、S、G2、M 4個時期，其中S期是DNA合成期，細胞內(nèi)DNA進行半保留復(fù)制，各種構(gòu)成DNA的原料摻入到DNA中。BrdU作為一種胸腺嘧啶核苷的類似物（其化學(xué)結(jié)構(gòu)特點是胸腺嘧啶的堿基嘧啶環(huán)上與5位C原子連接的甲基被溴代替），像胸腺嘧啶核苷一樣可摻入到細胞合成的DNA中。當細胞處于DNA合成期（S期）而同時又有BrdU存在時, 就會有BrdU摻入新合成的DNA中，只要細胞不消亡，這種BrdU就在胞核的DNA中長期存留。摻入到D

7、NA的BrdU可通過抗BrdU單克隆抗體在組織切片或細胞爬片上顯示。BrdU抗體比較大，由于DNA雙鏈結(jié)構(gòu)的位阻，BrdU抗體無法直接與雙鏈上的BrdU結(jié)合，必須先用使DNA部分變性，這樣變性了的DNA單鏈上的BrdU才能與BrdU抗體結(jié)合，因此做BrdU細胞增殖實驗一定要變性，當然變性的方法包括酸解，熱解等，但是要注意變性的程度也很重要。建議采用EdU細胞增殖檢測方法，無需變性，無需酶解，無需抗體，小分子染色，3小時完成實驗。EdU是一種胸腺嘧啶核苷類似物，能夠在細胞增殖時期代替T滲入正在復(fù)制的DNA分子，通過基于EdU與Apollo?熒光染料的特異性反應(yīng)檢測DNA復(fù)制活性，通 快速、更靈敏

8、、更準確。EdU檢測染料只有BrdU抗體大小的1/500，在細胞內(nèi)很容易擴散，無需DNA變性（酸解、熱解、酶解等）即可有效檢測，可有效避免樣品損傷，在細胞和組織水平能更準確地反映細胞增殖等現(xiàn)象。該方法能對細胞周期進行迅速而穩(wěn)定的測量, 而且標記BrdU的細胞只要不受到紫外線照射，對細胞本身沒有功能損害。該技術(shù)可應(yīng)用到跟蹤檢測移植細胞的存活、分化和功能狀態(tài)。3. DAPI染色原理DAPI為4，6二脒基-2-苯吲哚(4，6diamidino-2phenylindole)，能與雙鏈DNA小槽，特別是AT堿基結(jié)合，也可插入少于3個連續(xù)AT堿基對的DNA序列中。當它與雙鏈DNA結(jié)合時，熒光強度增強20倍

9、，而與單鏈DNA結(jié)合則無熒光增強現(xiàn)象，因此是一種簡易、快速和敏感地檢測DNA的方法。DAPI的熒光強度雖較Hoechst低，但熒光穩(wěn)定性優(yōu)于Hoechst；其特異性較溴化乙啶(ethidlium bromide，EB)和碘化丙啶(propidium iodide，P1)高。DAPI的中文名稱是4，6-聯(lián)脒-2-苯基吲哚，是一種常用的熒光染料，其作用機理與溴化乙錠（EB）等染色劑的機理類似：它們與DNA雙螺旋的凹槽部分可以發(fā)生相互作用，從而與DNA的雙鏈緊密結(jié)合。結(jié)合后產(chǎn)生的熒光基團的吸收峰是358nm而散射峰是461nm，正好UV（紫外光）的激發(fā)波長是356nm，使得DAPI成為了一種常用的熒

10、光檢測信號。ANALYSIS OF CELL CYCLE1. INTRODUCTION Cell 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

11、 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 Iodide The classical method for the analysis of cell cycle distribution is t

12、he 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 bin

13、d 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-molar

14、ity HCl for the denaturation of DNA. Furthermore, this method may be utilized either for unfixed or for fixed cells in suspension. B) Cyclins/Propidium Iodide Cyclins are key components of the cell cycle progression machinery. In particular, the expression of cyclins D, E, A and B1 provides new cell

15、 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 i

16、n early G1, the peak of cyclin E is typical of G1/S transition, the peak of cyclin A 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 ne

17、cessary 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 Iodide One of the most used protocol for the determination of apoptosis in the different phases of

18、 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 or

19、der to have more details, see the Chapters related to TUNEL technique. D) F-Actin/Propidium Iodide The 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 cytos

20、kleton 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 PROTOCOL A.2.1 Materials BrdU (A2), washing b

21、uffer (A1), HCl 4 M, Borax buffer (A1), anti-BrdU antibody (A2), goat-anti-mouse-FITC antibody (A2), PI buffer (A1). A.2.2. Methodology 1. 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

22、 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

23、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

24、 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 information In 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 B