1、基礎(chǔ)的細(xì)胞實(shí)驗(yàn)曲片曲片體外培養(yǎng)細(xì)胞一代生存期分為游離期、貼壁期、潛伏期、對(duì)數(shù)生長(zhǎng)期、停止期（平臺(tái)期）。對(duì)數(shù)生長(zhǎng)期：細(xì)胞數(shù)隨時(shí)間變化成倍增長(zhǎng)，活力最佳，細(xì)胞數(shù)量呈指數(shù)增長(zhǎng)，細(xì)胞群體 均一。最適合進(jìn)行實(shí)驗(yàn)研究。細(xì)胞搖勻的經(jīng)驗(yàn)：孔越小，越要好好搖。種的時(shí)候可以有意識(shí)的分散種細(xì)胞，而不是直接一個(gè)小區(qū)域種下 去。 96孔板種細(xì)胞輕點(diǎn)打進(jìn)孔里，打的太猛細(xì)胞容易聚集在邊緣。六孔板手動(dòng)8 字晃 勻效果比較好。種細(xì)胞時(shí)晃動(dòng)細(xì)胞很重要，但應(yīng)避免在桌子上推著前后左右晃。在手中 兩個(gè)方向的八字晃會(huì)更穩(wěn)更好。細(xì)胞計(jì)數(shù)細(xì) fJ LJ1ef -111fi DErfi OOBOTfH細(xì)胞懸液的細(xì)胞數(shù)/ml=（四個(gè)大格子細(xì)胞數(shù)

2、/4） X稀釋倍數(shù)x104/ml 計(jì)數(shù)建議:1）壓邊線(xiàn)細(xì)胞：計(jì)上不計(jì)下，計(jì)左不計(jì)右；2）鏡下偶見(jiàn)有兩個(gè)以上細(xì)胞組成的細(xì)胞團(tuán)，應(yīng)按單個(gè)細(xì)胞計(jì)算，若細(xì)胞團(tuán)10%以上， 說(shuō)明分散不好，需重新制備細(xì)胞懸液；3）每個(gè)細(xì)胞懸液至少滴樣兩次求平均值。提問(wèn)：細(xì)胞計(jì)數(shù)的濃度控制在多少？計(jì)數(shù)重復(fù)幾次？答：建議濃度控制在50-100萬(wàn)/ml。建議表型實(shí)驗(yàn)計(jì)數(shù)4次，普通實(shí)驗(yàn)計(jì)數(shù)2次。建議 全部計(jì)數(shù)完再一起種板。注意點(diǎn)：細(xì)胞計(jì)數(shù)不要同時(shí)記超過(guò)4株以上的細(xì)胞。若有需要，先消化記4株，細(xì)胞濃 度調(diào)好靜置一邊；再消化計(jì)另外的。而后按消化和計(jì)數(shù)順序種細(xì)胞。這樣可以避免多株 細(xì)胞同時(shí)消化而帶來(lái)的消化不理想。常用細(xì)胞培養(yǎng)器皿實(shí)驗(yàn)室

3、常用細(xì)胞培養(yǎng)扳和瓶皿HtccmmerMiKlMedium Volume HtccmmerMiKlMedium Volume ( ml JCdr neid9.5l.a34110*4CLja-c.sf輜恥fl?50-.1J-O2S5Mg3.21CP2如1曲抑D9*1J-2.7蟲(chóng)氈沖21IkrnWUlW瑋hl護(hù)255 5 j兀W亦弭tMKn1&-22SJi-UFHiSsBFFCORNlhlGSWfflill站丁亀尋血用液氮是低溫制品，在使用過(guò)程中要防止凍傷。在液氮中操作及存取冷凍物品時(shí)速度要快 要注意輕拿輕放，以免內(nèi)容物解凍，造成不必要的損失。5. 細(xì)胞傳代消化溫度：室溫或37C。消化時(shí)間：不超過(guò)1

4、0 min,也不可太短(須形成單細(xì)胞懸液)。A 注意點(diǎn)：防止細(xì)胞成片滑落(4C消化，延長(zhǎng)消化時(shí)間較易獲得單細(xì)胞懸液)；輕柔吹打，防止機(jī)械損傷；離心時(shí)不超過(guò)300 g (1000 rpm),實(shí)驗(yàn)室目前離心所用轉(zhuǎn)速為800rpm；盡量避免刮傷培養(yǎng)瓶細(xì)胞貼附面，否則影響觀(guān)察且細(xì)胞貼壁不均勻；及時(shí)換液和傳代，不可拖延，避免細(xì)胞過(guò)爆后細(xì)胞狀態(tài)不好。6. 細(xì)胞消化條件參考Cell line胰酶條件時(shí)間傳代比例長(zhǎng)滿(mǎn)時(shí)間10cm dish 細(xì)胞數(shù)Huh1EPET37 C4 min1： 45d400wHuh7EPET37 C2 min1:64d200wHLEEPET37 C3 min1:83d200wHLFEP

5、ET37 C3 min1:83d200wHepG20.25%Trypsin37 C4 min1:35d200wHep3B0.25%Trypsin37 C1.5 min1:44d-HUCCT10.25%Trypsin37 C6 min1:103d500wRBEEPET37 C2 min1:103d200-300wHuh280.25%Trypsin37 C6 min1:33d30w293T1/10 EPETRT1 min1:203d1000w3T3EPET37 C3 min1:83d400wLX2EPETRT1 min1:3-1:42-3d-THP1-1:43-7. 換液時(shí)機(jī)pH 降低。培養(yǎng)基顏色

6、由紅變橙要警惕，變成黃色前一定要換液。pH 降至 6.5 時(shí)，細(xì)胞停止生長(zhǎng)。pH 降至6.0時(shí)，細(xì)胞失去活性。2）發(fā)現(xiàn)細(xì)胞出現(xiàn)形態(tài)衰退時(shí)須勤換液。3）細(xì)胞密度過(guò)低或生長(zhǎng)緩慢，則更換一半培養(yǎng)基。細(xì)胞凍存（慢凍）預(yù)先配制凍存液：10%DMSO +細(xì)胞生長(zhǎng)液（50%血清+40%基礎(chǔ)培養(yǎng)液）。由于DMSO稀釋時(shí)會(huì)放出大量熱能，故不可將DMSO直接加入細(xì)胞液中，必須使用前 先行配制完成。取對(duì)數(shù)生長(zhǎng)期細(xì)胞,經(jīng)胰酶消化后，加入適量?jī)龃嬉?，用吸管吹打制成?xì)胞懸液（1-5x106 cell/ml）加1 ml細(xì)胞懸液于凍存管中，密封后標(biāo)記冷凍細(xì)胞名稱(chēng)、冷凍日期、代數(shù)、細(xì)胞數(shù)量 和實(shí)驗(yàn)者名字。液氮長(zhǎng)期保存。慢凍程序

7、：1）標(biāo)準(zhǔn)程序：采用細(xì)胞凍存器。當(dāng)溫度在-25C以上時(shí)，12 C/min當(dāng)溫度達(dá)-25C以下時(shí)，510 C/min當(dāng)溫度達(dá)-100C時(shí)，可迅速放入液氮中2）傳統(tǒng)程序：冷凍管置于4C 1 h -20C 1 h-80C16-18 h（或隔夜）T液氮槽長(zhǎng)期 儲(chǔ)存。細(xì)胞的復(fù)蘇方法（速融）注意點(diǎn)：37C水浴，快速解凍，避免慢速融化水分滲入細(xì)胞內(nèi)，再次形成胞內(nèi)結(jié)晶損傷 細(xì)胞。凍存細(xì)胞從液氮中取出后，立即放入37C水浴中，輕輕搖動(dòng)冷凍管，使其在1 min內(nèi)（不 要超過(guò)3 min）全部融化，5 min內(nèi)用培養(yǎng)液稀釋至原體積的10倍以上。兩種解凍后處理方法：1）解凍后的細(xì)胞直接接種到含完全生長(zhǎng)培養(yǎng)液的細(xì)胞培養(yǎng)皿

8、進(jìn)行培養(yǎng),24 h后更換培 養(yǎng)液，以去除 DMSO。2）解凍后的細(xì)胞先通過(guò)低速離心10 min去除冷凍保護(hù)劑，然后再接種到含完全生長(zhǎng) 培養(yǎng)液的培養(yǎng)皿中。熒光顯微鏡啟動(dòng)高壓汞燈后，不得在 30 min 內(nèi)將其關(guān)閉；關(guān)閉后，必須待汞燈冷卻后 方可再次打開(kāi)。Lentivirus production in 293T cellsUsing Lipofectamine 3000, Ji lab, 2019The day before transfection ( Day 1), passage1/3 10 cm dish 293T cells in a new 10cm dish so that the

9、y will be 70-80% confluent on the day of transfection.On the day of transfection (Day 2), remove the culture medium from the 293T cells and replace with 6 ml of fresh medium (without antibiotics) containing serum.For each transfection sample, prepare DNA-Lipoectamine 3000 complexes as follows: In a

10、sterile 1.5 ml tube with 0.5ml Opti-MEM, add:Packagi ng Plasmid-psPAX2En velope Plasmid-pMD2.G or(10703bp, addge ne12260)(5824bp, addge ne12259)5.3ug1.4ugPackaging Plasmid-pCMV-dR8.2 dvpr (13457bp, addgene8455)6.6ugEn velope Plasmid-pCMV-VSV-G(6363bp, addge ne8454)1.6ugANDTransfer Plasmid-Lenti-miR/

11、miRZip-antimiR (SBI, 7.5/7.9kb) 5.3ugNote:The proper molar ratio shall be Envelope Plasmid：Packaging Plasmid： Transfer Plasmid=1:2:34.Adding p3000. p3000 (volume): plasmid (ug) =2:1Mix gently, RT for 5 mins.In a separate sterile 1.5 ml tube with 0.5ml Opti-MEM, add: Lipofectamine 3000 20ul.Mix gentl

12、y, RT for 3-5 mins (Note: has to be less than 15mins)After the incubation, combine the above diluted DNA with the diluted Lipofectamine 3000.Mix gently. Incubate, RT for 20 minutes.Add the DNA-Lipofectamine3000 complexes to each dish of cells. Mix gently by rocking the plate back and forth.After 24

13、hours post transfection (Day 3), add 8 ml fresh medium (without antibiotics) containing serum. Incubate at 37C in a humidified 5% CO2 incubator.Harvest virus-containing supernatants 52hours posttransfection (Day 4) by remov ing medium into to a 15 ml sterile tube, keep on ice.Centrifuge supernatants

14、 at 2000 rpm for 10 minutes at +4C to pellet debris.Filter the viral supernatants through a 0.45 |im filter.Aliquot viral supernatants into 1.5 ml tubes (0.5ml/tube). Store viral stocks at -80C.Proceed to Titer Your Viral Stock.Note: If use lipo 2000, no need add p3000. If use PEI 40,000, PEI: plasm

15、id=1.875: 1Titer LentiVirusViacounting GFPcells, Ji lab, 2016The day before transduction (Day 1), trypsinize and count the 3T3 cells, plating 3000 cells/well of 96-well plate. Incubate cells at 37C overnight in a humidified 5% CO2 incubator.On the day of transduction (Day 2), thaw your Lentiviral st

16、ock and prepare 10-fold serial dilutions ranging from 10-1 to 10-8. For each dilution, dilute the Lentiviral stock into complete culture medium to a final volume of 0.15 ml.DO NOT vortex, But mix well.Using 96-well plate to do the dilution and tittering.123456789101112ADDDDDD3T33T33T33T33T33T3BDDDDD

17、D3T33T33T33T33T33T3CDDDDDD3T33T33T33T33T33T3DDDDDDD3T33T33T33T33T33T3EDDDDDD3T33T33T33T33T33T3FDDDDDD3T33T33T33T33T33T3GDDDDDD3T33T33T33T33T33T3HDDDDDD3T33T33T33T33T33T3Column #1-#6 is used for dilution.Add 135 ul of culture medium to each dilution well. Line A: add 15 ul virus from original lentivi

18、rus stock. Mix well Line B: add 15 ul virus from the well of line A. Mix well Line C: add 15 ul virus from the well of line B. Mix well So, line A is 10 x dilution; line B is 100 x dilution.Remove the culture medium from the cells. Mix each dilution gently by pipetting and add 0.1 ml to one well of

19、cells (total volume = 0.1 ml).Add Polybrene to each well to a final concentration of 8 |ig/ml.Swirl the plate gently to mix. Incubate at 37C overnight in a humidified 5% CO2 incubator.The following day (Day 3), remove the media containing virus and replace with0.1 ml of complete culture medium. Incu

20、bate at 37C overnight in a humidified 5% CO2 incubator.Incubate cells for an additional 3 days. Analyze the percentage of GFP-positive cells.Calculate the titer (TU/ml) by with the formula:Titer = # of positive clones / 0.1ml x times of dilution腺相關(guān)病毒(Adeno-Associated Viral Vector, AAV)腺相關(guān)病毒屬微小病毒科(pa

21、rvovirus),為無(wú)包膜的單鏈線(xiàn)狀DNA病毒。AAV的基因組 約4700bp,包括上下游兩個(gè)開(kāi)放讀碼框架(ORF),位于分別由145個(gè)核苷酸組成的2個(gè)反 向末端重復(fù)序列(ITR)之間?；蚪M中有3個(gè)啟動(dòng)子(P5、P19和P40)和2個(gè)開(kāi)放閱讀讀框(ORF), rep和cap, 如圖所示。rep編碼4個(gè)重疊的多功能蛋白，即Rep78、Rep68、Rep52和Rep40,其中 Rep78 與 Rep68 參與 AAV 的復(fù)制與整合, Rep52 和 Rep40 具有解螺旋酶和 ATP 酶活性, 與Rep78、Rep68共同參與單鏈基因組的復(fù)制；cap編碼的VP1、VP2、VP3是裝配成完 整病毒所需要的衣殼蛋白,它們?cè)?AAV 病毒整合、復(fù)制和裝配中其重要作用。From 腺相關(guān)