养个细胞咋就还发生爆炸了呢？
本帖最后由 屈泰龙 于 2016-4-11 18:53 编辑https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7knQThYfhnRupibb5OMvQ8qHUX0Jrc3gVNsTSoRvsM7ndkhxIVcUfE8pDHUyOiaKMiaLaOzA1MvAhO6Q/0
昨天，zhenglinlng05坛友估计踏踏实实的被惊到了，复苏细胞时发生了file:///C:/Users/x240/AppData/Local/Temp/msohtmlclip1/01/clip_image002.jpgfile:///C:/Users/x240/AppData/Local/Temp/msohtmlclip1/01/clip_image003.jpgfile:///C:/Users/x240/AppData/Local/Temp/msohtmlclip1/01/clip_image004.jpg https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhlVn1AiamQD8eobFKMQkbvXwsO9VJo2JicypucLTiaeCd2Ydrnyibn6RdJdw/0?wx_fmt=jpeg https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhlPBsEpxdYnoibF2Og3FnKvdDDTzuNG3xH8p2qy7icVNRQRnRzpSV1wKZQ/0?wx_fmt=jpeg https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhlQPgHE3HqibtEatuAXxKucjeGdaOibCic7nTlichNibKo8qXMBaVwLCYLzibQ/0?wx_fmt=jpeg
，
嗯好吧，其实没那么恐怖，是小编夸张了那么一点啦。但我估计当时这位坛友是这样的表现？https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhlb51aH9tZHkHFYUqm2Hia4jzsH5ZNydibHkazzvDQpGxsTzzDkTicY3ycg/0?wx_fmt=jpeg，嘿嘿。。。
来看一下这位坛友的帖子：https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhlW679G0ZUibontxu93jwKY5vI3ZatHFNNaiaJHKleAdMfFOFMVerblNvQ/0?wx_fmt=jpeghttps://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhlVRP0frXUsicEratyevs2VaGqXzubuMp1J4S1ZtN4N7evxZgAgZonwsw/0?wx_fmt=jpegfile:///C:/Users/x240/AppData/Local/Temp/msohtmlclip1/01/clip_image009.png回复是这样的。。。
https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhlrh2pQ3TvFJ38piaD7DqqBeJiaxw9PZIiberrnN1CUHjylYqEcCMXRJOPg/0?wx_fmt=pngfile:///C:/Users/x240/AppData/Local/Temp/msohtmlclip1/01/clip_image011.jpg和这样的。。。
https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhl5fG78ppBVyRUHCgPzVv9yJpibKmn1MOrh60dpWsLpdXFmWWK5cSEepA/0?wx_fmt=pngfile:///C:/Users/x240/AppData/Local/Temp/msohtmlclip1/01/clip_image013.jpg有没有觉得他们的回答很专业？https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhlzUibPnZUPp38hIZPTTzibicXUdh6pv1IRhKI9q49rz2ic6gmicX03wKMvibw/0?wx_fmt=gif
那是别人说的，你的呢？你的呢？你的呢？那我们来看一下Culture of Animalcells :A MANUAL OF BASIC TECHNIQUE 是怎么做得。
细胞冻存PROTOCOL FREEZING CELLSOutlineGrow the cultureto late log phase, prepare a high concentration cell suspension in medium witha cryoprotectant , aliquot into ampoules, and freeze slowly.https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhlP8wqbfGeQntpvRMtJDtLUEODysybVr7EU2xrbCQficwo4Vf7EJAJyHg/0?wx_fmt=png
file:///C:/Users/x240/AppData/Local/Temp/msohtmlclip1/01/clip_image015.jpgMaterialsSterile or AsepticallyPrepared：Culture to be frozenIf monolayer: PBSA and0.25% crude trypsin，Growth medium ：Serumimproves survival of the cells after freezing; up to 50%, or even pure, serumhas been used. If serum is being used with serum-free cultures, it should bewashed off after thawing.Cryoprotectant , free ofimpurities: DMSO in a glass or polypropylene vial, or glycerol, fresh and in auniversal containerSyringe, 1–5 mL, fordispensing glycerol (because it is viscous)Plastic ampoules, 1.2 mL,prelabeled with the cell line designation and the date of freezingNonsterile: Hemocytometeror electronic cell counterCanes or racks forstorage (racks may already be in place in the freezer)Insulatedcontainer for freezing: polystyrene box lined with cotton wool or plastic foaminsulation tube。
Protective gloves,nitrileProtocol1. Make sure theculture satisfies the criteria for freezing (see Table below) and check by eyeand on microscope for:(a) Healthy appearance (b) Morphologicalcharacteristics https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhld6U3XCrdpOFJrXbE236AwJxMnCpLSbxE9vDNDl8kzrmSZhibmrXgzSQ/0?wx_fmt=jpeg (c) Phase ofgrowth cycle (should be late log phase before entering plateau 对数生长期，时间也很关键呀(d) Freedom fromcontamination 无污染，要纯洁呀同学file:///C:/Users/x240/AppData/Local/Temp/msohtmlclip1/01/clip_image021.jpghttps://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhlRQvI5je1BEHWVibHHSDCOmydFfic4AMA7hUsOuGUwQib185jUiaicEzGMFg/0?wx_fmt=png
2. Grow theculture up to the late log phase and, if you are using a monolayer, trypsinizeand count the cells. If you are using a suspension, count and centrifuge thecells.3. Resuspend at 2× 106–2 × 107cells/mL.4. Dilute one ofthe cryoprotectants in growth medium to make freezing medium:(a) Add dimethylsulfoxide (DMSO) to 10–20%file:///C:/Users/x240/AppData/Local/Temp/msohtmlclip1/01/clip_image022.png∆ SafetyNote. DMSO can penetrate many synthetic and naturalmembranes, including skin and rubber gloves. Consequently, any potentiallyharmful substances in regular use (e.g., carcinogens) may well be carried intothe circulation through the skin and even through rubber gloves. DMSO shouldalways be handled with caution, particularly in the presence of any toxicsubstances.https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhl4UTc1IMib7V0AApI6bDK0x5z9L75lNH9JRu4KCZr3kuyaprMPLSR2qA/0?wx_fmt=jpeg
Or(b) Add glycerolto 20–30%.5. Dilute the cellsuspension 1:1 with freezing medium to give approximately 1 × 106–1 × 107cells/mL and 5–10%DMSO (or 10–15%glycerol). It is notadvisable to place ampoules on ice in an attempt to minimize deterioration ofthe cells（不要自作聪明哦）. A delay of up to 30 min at room temperatureis not harmful when using DMSO and is beneficial when using glycerol.6. Dispense thecell suspensions into prelabeled ampoules, and cap the ampoules with sufficienttorsion to seal the ampoule without distorting the gasket.7. Place theampoules on canes for canister storage, or leave them loose for drawer storage.8. Freeze theampoules at 1◦C/min by one of the methods described below。With the insulatedcontainer methods, this will take a minimum of 4–6 h after placing themat −70◦C if starting from a 20◦C ambient temperature .but preferably leave the ampoules in the containerat −70◦C overnight.程序很重要啊，多看两遍吧！https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhlghgQSmkicyGiaQQ5iahrCtciaAf1AHQKu7EJ7QSChXLLd0933I8yJDWdKw/0?wx_fmt=png
file:///C:/Users/x240/AppData/Local/Temp/msohtmlclip1/01/clip_image023.png9. When theampoules have reached −70◦C or lower, check the freezer record before removing the ampoulesfrom the −70◦C freezer or controlledrate freezer, and identify a suitable location for the ampoules.10. Transfer theampoules to the liquid N2 freezer, preferably not submerged in the liquid,placing the cane and tube into the predetermined canister or individualampoules into the correct spaces in the predetermined drawer. This transfermust be done quickly (<2 min), as the ampoules will reheat at ∼10◦C/min, and the cells will deteriorate rapidly ifthe temperature rises above −50◦C.∆ Safety Note. Protectivegloves and a face mask should be used when placing ampoules in liquid nitrogen.11. When theampoules are safely located in the freezer, complete the appropriate entries inthe freezer index.
你以为这就完啦？https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhl6seIsr4RML2CRLPHdBFkzKrRjGiabicwCia8BibdqTWmfsXRxgAia31xyfA/0?wx_fmt=png
细胞冻存之后还要进行复验，确认细胞冻存成功，好吧，一大波words 正在coming，准备接招。。。细胞复苏PROTOCOL 20.2.THAWING FROZEN CELLSOutlineThaw the cells rapidly, dilute them slowly, and reseed them at ahigh cell density.（快融缓吹高密度，）https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhl8lajfTzgEENB17fDZbrubflu3XzN94gQf5YHwzgNR4mD5wDrVUnU6g/0?wx_fmt=png
MaterialsSterile:Culture flaskCentrifuge tube(if centrifugation is required)Growth mediumPipettes, 1 mL, 10mLSyringe and 19-gneedle (if you are using glass ampoules)Nonsterile:Protective glovesand face maskSterile water at37◦C, 10 cm deep in a clean, alcohol-swabbed bucket with lidForceps：Alcohol, 70%SwabsNaphthalene Black(Amido Black), 1%, or Trypan Blue, 0.4%.Protocol1. Check the indexfor the location of the ampoule to be thawed.2. Collect allmaterials, prepare the medium, and label the culture flask.3. Retrieve theampoule from the freezer, check from the label that it is the correct one, and,if it has not been submerged in liquid nitrogen, place it in sterile water at37◦C in a beaker, plastic bucket, or water bath. If possible, avoidgetting water up to the cap as this will increase the chance of contamination.file:///C:/Users/x240/AppData/Local/Temp/msohtmlclip1/01/clip_image027.png∆ SafetyNote.Aclosed lab coat and gloves must be worn when removing the ampoule from thefreezer. If the ampoule has been stored submerged in liquid nitrogen, a face shield, or protective goggles,as well as a closed lab coat and gloves, must be worn. Ampoules, includingplastic ampoules, stored in the liquid phase may inspire the liquid nitrogenand, on thawing,will explode violently. In this case a plastic bucket with a lid must be used for thawing to contain any explosion. https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhlFkS74cH4U4chPj6ib49uxrcdZI6V6CXsxRR8wGkepsaSHJkuHOia2zqQ/0?wx_fmt=jpeg
4. When theampoule has thawed, double-check the label to confirm the identity of thecells; then swab the ampoule thoroughlywith 70% alcohol, and open it in a laminar-flow hood.5. Transfer thecontents of the ampoule to a culture flask with a 1-mL pipette.6. Add mediumslowly to the cell suspension: 10 mL over about 2 min added dropwise at thestart, and then a little faster, gradually diluting the cells and cryoprotectant.For cells that require centrifugation to remove the cryoprotectant:(a) Dilute thecells slowly, as in Step 6, but in a centrifuge tube or universal container.(b) Centrifugethem for 2 min at 100 g.(c) Discard thesupernatant medium with the cryoprotectant.(d) Resuspend thecells in fresh growth medium.(e) Seed flask forculture.7. The dregs inthe ampoule may be stained with Naphthalene Black or Trypan Blue to determine theirviability.8. Check after 24h:(a) For attached monolayercells, confirm attachment and try to estimate percentage survival based onphotographs of cells at the expected density (cells/cm2 ) with full survival.(b) For suspension-growingcells, check appearance (clear cytoplasm, lack of granularity), and dilute toregular seeding concentration. This can be made more precise if the cells arecounted and an estimate of viability is made, in which case the cells can bediluted to the regular seeding concentration of viable cells.https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhlqmAra8D02f7rJiafoRCReK60XHgvzQXVn4ypPgZ3eMdOovaGrPE37Eg/0?wx_fmt=png
看不懂？为啥没有中文版？因为，https://mmbiz.qlogo.cn/mmbiz/c10u0hdaD7mwSXqqwo8TyPRwHaVrFibhl59Vko9ofRYGBSibzaba0yZ3jWsqibkTlLQqj3zgOFxqhI7HKhsRBOIew/0?wx_fmt=jpeg
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