Brdu細(xì)胞增殖檢測試驗(yàn)

1、brdu 檢測細(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 萬個(gè)，在37、5%co字箱中培養(yǎng)72h（細(xì)胞密度2 至 50-60%左右）。2 . brdu （5-濱-2 -脫氧尿音）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：0.1m

2、ol/l的硼酸鈉（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封閉，室溫1h,可在搖床上晃動。9 . 吸出bsa用pbs洗3次，每次5min。10 .力口一抗（尿口密噬脫氧核昔brdu （鼠單抗）1:200 ）,用1%bsa&釋，4度過夜。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%

3、bs麻釋，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dapi染細(xì)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pbsw釋），避光室溫反應(yīng)10min。0 10min次，每次3洗pbs染好的細(xì)胞爬片用 dapi.將15.16 .中性樹膠封片，熒光顯微鏡觀察，200x鏡下取5個(gè)視野，計(jì)數(shù)brdu陽性細(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dms沖，儲存濃度 為 100mg/ml 分裝，每管120ul ，

4、 -20 保存。b. 2 mol/l hcl :取 8.333ml 12 mol/l hcl 的濃 hcl,加入 ddwfe容至 50 ml。c. 0.1 mol/l 硼酸鈉：稱量 1.907g 硼砂（nab- 10ho 381.36 g/mol ）,力口 入 ddw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 pbsw釋 3%bsas 1%

5、g. 4%fps： 4%多聚甲醛。我是用ddwe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(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)胞膜的通透性大大使得一部分膜蛋白變性， 從而使通透性

6、進(jìn)一步加強(qiáng)。 triton-x-100 增加，并且利用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 區(qū)域，利用二抗連接不同的 證抗體識別的特異性 。二抗可以特異性識別一抗的 fc 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dna4、 m 個(gè)時(shí)期，其中s期是dn的成期，細(xì)胞內(nèi)細(xì)月ft增殖周期包括g1、s、g2作為一種胸腺喀噬核昔brdu進(jìn)行半保留復(fù)制，各種構(gòu)成dna勺原料摻入到dna中。原子連接的甲基被c 的類似物（其化學(xué)結(jié)構(gòu)特點(diǎn)是胸

7、腺嘧啶的堿基嘧噬環(huán)上與5位合成中。當(dāng)細(xì)胞處于 dna臭代替），像胸腺口密噬核昔一樣可摻入到細(xì)胞合成的dna中，只要細(xì)胞摻入新合成的 dnabrdu在時(shí)，就會有brdu期）而同時(shí)又有期（sbrdubrdu可通過抗摻入到 dna中長期存留。dna勺不消亡，這種brdu就在胞核的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dnak鏈結(jié)構(gòu)白位阻，brdu抗體無法直接與雙鏈上的brdu結(jié)合，必須先用使 dnab分變性，這本變性了的dna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。

8、建議采用edu細(xì)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(shí)完成實(shí)驗(yàn)。edu是一種胸腺口密噬核昔類似物，能夠在細(xì)胞增殖時(shí)期代替t滲入正在復(fù)制的dna#子，通過基于 edu與apollo?熒光染料的特 異性反應(yīng)檢測dnas制活性，通 快速、更靈敏、更準(zhǔn)確。edu檢測染料只 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(xì)胞和組織水平能更準(zhǔn)確地反映細(xì)胞增殖等現(xiàn)象。的細(xì)胞只要不受 brdu 該方法能對細(xì)胞周期進(jìn)行迅速而穩(wěn)定的測量, 而且標(biāo)記到紫外線照射，對細(xì)胞本身沒有功能損害。該技術(shù)可應(yīng)用到跟蹤檢測

9、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 ， 6 diamidino-2 phenylindole) ，能與雙鏈dnam亂牛別是at堿基結(jié)合，也可插入少于 3個(gè)連續(xù)at堿基對的dnaf列中。當(dāng)它與雙鏈 dna吉合時(shí)，熒光強(qiáng)度增強(qiáng) 20倍，而與單鏈dna吉合則無熒光增強(qiáng)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(qiáng)度雖較hoechst 低，但熒光穩(wěn)定性優(yōu)于 hoechst ；其特異性較漠化乙呢(ethidlium bromide , eb)和碘化丙噬(propidium iodide , p1)高。

10、dapi的中文名稱是4, 6-聯(lián)瞇-2-苯基口引噪，是一種常用 的熒光染料，其作用機(jī)理與漠化乙錠(eb)等染色劑的機(jī)理類似：它們與 dnaz螺旋的凹槽部分可以發(fā)生相互作用，從而與dna勺雙鏈緊密結(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)波長是356nm,使得dapi成為了一種常用的熒光檢測信號。analysis of cell cycle1. introductioncell cycle and apoptosis are very important functional parameters toassess the cellular m

11、etabolism, 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 phasesapoptosisand cycle cell of assessme

12、nt 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 (brdu). the procedurerequ

13、ires 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 fluorescence from thefluorescein-

14、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.b) cyclins/propidium iodi

15、decyclins 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 detectable usingspecific

16、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 typical of late g2/m.using this

17、 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, evenif in avery few cell

18、 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 for the incorporation offlu

19、orescein-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 cyclespecificity is also p

20、ossible 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, are used. furthermore, this

21、 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 buff

22、er 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 4 c in the dark.10. as in step

23、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. incubate for 15-30 min at 416. analyse with flow

24、 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 used.