Brdu細(xì)胞增殖檢測(cè)實(shí)驗(yàn)行業(yè)材料

1、brdu檢測(cè)細(xì)胞增殖實(shí)驗(yàn)實(shí)驗(yàn)操作:1 鋪細(xì)胞，每個(gè)3.5cm dish 10萬(wàn)個(gè)，在37、5%co2孵箱中培養(yǎng)72h（細(xì)胞密度至50-60%左右）。2 brdu（5-溴-2-脫氧尿苷）加入培養(yǎng)細(xì)胞中，1mg/ml，標(biāo)記48h。(量：brdu以鋪滿整個(gè)dish底面為準(zhǔn)。)3 固定：pbs洗細(xì)胞爬片3次，每次5min，在搖床上晃動(dòng)清洗，4%pfa固定30min。4 變性：將固定好的細(xì)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(dòng)。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(guò)夜。11將孵好一抗的細(xì)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(xì)胞爬片用pbs洗3次，每次10min。14加dap

3、i染細(xì)胞核，儲(chǔ)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(xì)胞爬片用pbs洗3次，每次10min。16中性樹(shù)膠封片，熒光顯微鏡觀察，200×鏡下取5個(gè)視野，計(jì)數(shù)brdu陽(yáng)性細(xì)胞和藍(lán)染的細(xì)胞核數(shù)目，然后進(jìn)行統(tǒng)計(jì)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(chǔ)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(lái)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(dān)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(shí)搖晃混勻，16-24小時(shí)pfa會(huì)完全溶解。補(bǔ)充ddw，調(diào)節(jié)ph值。實(shí)驗(yàn)原理：1. 免疫染色實(shí)驗(yàn)的基本原理利用固定劑（通常是甲醛或多聚甲醛）將細(xì)胞固定，使得細(xì)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(jìn)一步加強(qiáng)。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(dòng)力學(xué)的關(guān)系可以通過(guò)競(jìng)爭(zhēng)性的反應(yīng)與目的蛋白結(jié)合，這一過(guò)程可以保證抗體識(shí)別的特異性

6、。二抗可以特異性識(shí)別一抗的fc區(qū)域，利用二抗連接不同的熒光基團(tuán)，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(dá)情況。2. brdu標(biāo)記原理細(xì)胞增殖周期包括g1、s、g2、m 4個(gè)時(shí)期，其中s期是dna合成期，細(xì)胞內(nèi)dna進(jìn)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(diǎn)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(xì)胞合成的dna中。當(dāng)細(xì)胞處于dna合成期（s期）而同時(shí)又有brdu存在時(shí), 就會(huì)有brdu摻入新合成的dna中，只要細(xì)胞不消亡，這種brdu就在胞核的dna中長(zhǎng)期存留。摻入到d

7、na的brdu可通過(guò)抗brdu單克隆抗體在組織切片或細(xì)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(wú)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(xì)胞增殖實(shí)驗(yàn)一定要變性，當(dāng)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(xì)胞增殖檢測(cè)方法，無(wú)需變性，無(wú)需酶解，無(wú)需抗體，小分子染色，3小時(shí)完成實(shí)驗(yàn)。edu是一種胸腺嘧啶核苷類似物，能夠在細(xì)胞增殖時(shí)期代替t滲入正在復(fù)制的dna分子，通過(guò)基于edu與apollo?熒光染料的特異性反應(yīng)檢測(cè)dna復(fù)制活性，通 快速、更靈敏

8、、更準(zhǔn)確。edu檢測(cè)染料只有brdu抗體大小的1/500，在細(xì)胞內(nèi)很容易擴(kuò)散，無(wú)需dna變性（酸解、熱解、酶解等）即可有效檢測(cè)，可有效避免樣品損傷，在細(xì)胞和組織水平能更準(zhǔn)確地反映細(xì)胞增殖等現(xiàn)象。該方法能對(duì)細(xì)胞周期進(jìn)行迅速而穩(wěn)定的測(cè)量, 而且標(biāo)記brdu的細(xì)胞只要不受到紫外線照射，對(duì)細(xì)胞本身沒(méi)有功能損害。該技術(shù)可應(yīng)用到跟蹤檢測(cè)移植細(xì)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(gè)連續(xù)at堿基對(duì)的dna序列中。當(dāng)它與雙鏈dna結(jié)合時(shí)，熒光強(qiáng)度增強(qiáng)20倍

9、，而與單鏈dna結(jié)合則無(wú)熒光增強(qiáng)現(xiàn)象，因此是一種簡(jiǎn)易、快速和敏感地檢測(cè)dna的方法。dapi的熒光強(qiáng)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(jī)理與溴化乙錠（eb）等染色劑的機(jī)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(tuán)的吸收峰是358nm而散射峰是461nm，正好uv（紫外光）的激發(fā)波長(zhǎng)是356nm，使得dapi成為了一種常用的熒

10、光檢測(cè)信號(hào)。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 brdu