细胞培养实验指导大全共37页文档
细胞培养实验步骤大全(精华版)
细胞培养实验步骤大全(精华版)细胞培养是生物学研究中常用的技术，在许多实验室中都扮演着重要角色。
下面是细胞培养的一般步骤，供参考。
材料准备1. 细胞培养基：根据实验需求选择适当的培养基，如DMEM、RPMI 1640等。
2. 细胞培养器具：培养皿、离心管、移液器等。
3. 细胞培养室：确保环境清洁、无菌。
细胞准备1. 细胞来源：选择要培养的细胞系或原代细胞。
2. 细胞传代：如果使用的是细胞系，根据需要进行传代，确保细胞状态良好。
细胞培养1. 细胞计数：使用显微镜和细胞计数仪等工具，准确计算细胞数目。
2. 细胞接种：按照实验要求，在含有培养基的培养皿中接种适量的细胞。
根据细胞类型的不同，可能需要预先涂覆培养皿。
3. 细胞培养条件：根据细胞类型的要求，提供适宜的培养条件，如温度、湿度和CO2浓度等。
4. 培养培养：定期更换培养基，保持细胞处于良好的生长状态。
5. 细胞观察：使用显微镜观察细胞形态和生长情况，检测是否存在异常。
细胞处理1. 细胞传代：根据细胞数量和状态，决定是否进行细胞传代。
传代可以延续细胞的寿命和维持细胞应答。
2. 细胞刺激：根据实验设计，给予细胞适当的刺激，如添加药物、因子或介质等。
3. 细胞采集：当需要获取细胞样本时，使用细胞剥离剂或胰蛋白酶等方法将细胞从培养皿中采集。
4. 细胞冻存：如果需要长期保存细胞，可以使用液氮冷冻保存的方法。
先加入冻存液，然后将细胞在适当条件下冷冻保存。
以上是细胞培养的一般步骤，根据具体实验和细胞类型，步骤可能会有所调整和细化。
在进行细胞培养实验时，请始终注意无菌操作和合理使用实验工具，以确保实验结果的准确性和可靠性。
细胞培养实验指导
细胞培养实验指导第五章细胞培养实验实验一普通光学显微镜的结构和使用【实验目的】1.熟悉显微镜的结构和各部件性2.掌握低倍镜、高倍镜的正确使用油镜的使用方法。
【实验内容】1.显微镜成像原理（图1―1）标本（F1）置于聚光器与物镜之间，镜、聚光器各自相当于一个凸透光线自反射镜折射入聚光器，经聚增强，照射在标本上。
标本的像经成像于F2处，像是倒像。
目镜将此步放大，并成像于人眼的视网膜上(F3)（正像）。
2.显微镜的结构图1-1显微镜成像原理能。
方法，熟悉目镜、物镜。
平行的光器集聚物镜放大例像进一图1-2 光学显微镜结构示意图1.目镜2.镜筒3.物镜转换器4.物镜5.透光孔6.聚光器7.光圈8.反光镜9.粗调节器10.细调节器 11.镜臂 12.移片器 13.载物台 14.倾斜关节 15.镜柱 16.镜座 17.照明1）机械部分（1）镜座：显微镜的基座。
起稳定和支持整个镜身的作用。
有的显微镜在镜座内装有照明光源等构造（图1―2）。
（2）镜臂：支持镜筒和镜台。
镜筒直立式光镜在镜臂和镜柱之间有一可活动的关节叫倾斜关节，可使镜臂作适当倾斜，便于观察。
镜筒倾斜式显微镜由于镜臂和镜柱连为一体，故无此关节。
（3）镜筒：位于镜臂前方的圆筒，上端安装目镜，下端装有旋转盘。
根据镜筒的数目，光镜可分为单筒式和双筒式两类。
（4）载物台：在镜筒下方，方形或圆形．放玻片标本用。
载物台中央有一圆形通光孔，两旁备有一压片夹。
有的载物台上装有标本移动器，移动器上装有弹簧夹，用于固定标本片。
移动器的一侧有两个旋钮，转动旋钮可使玻片前后左右移动。
（5）物镜转换器：圆盘状，在镜筒下方，其上装有3―4个放大倍数不同的物镜。
旋转物镜转换器可更换物镜。
（6）聚焦调节器；为调节焦距之用。
大旋钮为粗调节器，转动粗调节钮可使镜筒(或载物台)升降，调节焦距。
旋转一周可使镜筒(或戴物台)升降10mm。
一般用于低倍镜调焦。
小旋钮为细调节器，转动细调节钮可使镜筒(或超物台)缓慢升降，每旋转一周约使镜筒(或载物台)升降0.1mm。
医学研究中细胞培养技术实验指南
医学研究中细胞培养技术实验指南细胞培养技术是医学研究中不可或缺的重要手段之一，它为研究人类疾病、药物筛选和治疗方案提供了有力的工具。
本文将为您介绍细胞培养技术实验的指南，帮助您更好地开展医学研究中的细胞培养实验。
一、细胞培养基准备细胞培养基是细胞生长和繁殖所必需的基础培养环境，正确配制培养基是实验成功的关键。
1.1 选择合适的培养基根据所研究的细胞类型选择适合的培养基，通常，最常用的培养基有DMEM、MEM和RPMI 1640等。
另外，根据实验需要，可以添加胎牛血清、人血清、抗生素和生长因子等。
1.2 培养基的配方与储存根据细胞类型和实验需要，制备培养基的配制液，遵循供应商提供的配方和使用说明。
配制好的培养基应储存在4℃冰箱中，避免阳光直射，保质期根据供应商指引执行。
二、细胞培养槽和培养器具准备保持实验环境的清洁和无菌是细胞培养实验中的另一个关键点。
2.1 准备培养槽和培养器具将所需培养槽、培养瓶、离心管、吸头、培养皿、离心机等培养器具在高压蒸汽灭菌锅或培养箱中进行高温高压灭菌。
培养槽在灭菌后应放置在无尘台上，注意避免污染。
2.2 储备适量的试剂和培养物品体外培养需要用到的培养基、胎牛血清、人血清、生长因子、抗生素等试剂必须事先准备好，并按照实验操作规范使用。
三、细胞的分离和培养细胞的有效分离和培养对保证实验结果的准确性至关重要。
3.1 细胞的分离根据所要研究的细胞类型选择合适的方法进行细胞的分离，如胰蛋白酶消化法、组织弹性体分离法以及银离子胶体沉淀分离法等。
在进行细胞分离前，务必将所有操作具备无菌条件，避免外界环境的污染。
3.2 细胞的培养将分离得到的细胞用预先准备好的培养基进行悬浮培养。
培养器具需先预先灭菌处理，避免细胞感染。
培养皿或培养瓶中应添加适量的培养基，根据实验需要添加相应的血清、生长因子等。
将需要培养的细胞分散均匀地加入培养器具中，将培养器具放入恒温培养箱中，维持适当的温度、湿度和二氧化碳浓度。
实验二、细胞培养的准备共36页
61、辍学如磨刀之石，不见其损，日 有所亏 。 62、奇文共欣赞，疑义相与析。
63、暧暧远人村，依依墟里烟，狗吠 深巷中 ，鸡鸣 桑树颠 。 64、一生复能几，倏如流电惊。 65、少无适俗韵，性本爱丘山。
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26、要使整个人生都过得舒适、愉快，这是不可能的，因为人类必须具备一种能应付逆境的态度。——卢梭
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27、只有把抱怨环境的心情，化为上进的力量，才是成功的保证。——罗曼·罗兰
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28、知之者不如好之者，好之者不如乐之者。——孔子
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29、勇猛、大胆和坚定的决心能够抵得上武器的精良。——达·芬奇
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30、意志是一个强壮的盲人，倚靠在明眼的跛子肩上。——叔本华
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细胞培养操作指南
细胞培养操作指南1，原代细胞培养原代培养需要严格要求：（1）取材时需要去除脂肪和坏死的组织(2)为减少对组织的损伤，需用锋利的器具(3)适度离心以去除用于解离的酶（4）由于原代培养组织细胞的存活率很低，故用于原代培养的细胞的密度应高于正常的传代培养的细胞密度。
（5）营养丰富的培养基比单一的培养基更可取，如果要添加血清，胎牛血清比牛马的血清更好。
(6)对于取材部位易于感染（如皮肤）应先用70%乙醇消毒，在无菌条件下切除组织，尽快转移入BSS或培养基中。
不要再培养室取材，因为动物可引入微生物污染。
若必须推迟，可保存在4℃，72h。
1. 剪切组织先将所取得的组织，用D-Hanks或Hanks液清洗，以去除表面血污，并用手术镊去除粘附的结缔组织等非培养所需组织。
再次清洗后，用手术剪将组织剪成若干小块，移入青霉素小瓶或小烧杯中，加入适量缓冲液，用弯头眼科剪，反复剪切组织，直到组织成糊状，约1mm3大小。
静置片刻后，用吸管吸去上层液体，加入适当的缓冲液再清洗一次。
2. 消化分离消化分离的目的是将细小的组织块消化分离成细胞团或分散的单个细胞，以利于进一步的培养，常用的消化酶有胰蛋白酶和胶原酶。
3. 培养细胞悬液用计数板进行细胞计数，用培养液将细胞数调整为2～5×105 cells／m1，或实验所需密度。
分装于培养瓶中，使细胞悬液的量以覆盖后略高于培养瓶底部为宜。
置CO2培养箱内，5％CO2，37℃静置培养。
一般3～5d，原代培养细胞可以粘附于瓶壁，并伸展开始生长，可补加原培养液量1／2的新培养液，继续培养2～3 d后换液，一般7～14 d可以长满瓶壁，进行传代。
注意事项：1. 无菌操作细菌或霉菌污染是培养失败的常见原因，必须加强各个环节的无菌操作观念，以预防为主，一旦污染，一般是很难消除。
2. 培养液所用的培养液必须满足细胞生存和生长的必要条件。
由于细胞来源的动物种类、组织类型不同，对培养液的要求有一定的差异，必要时可用预实验的方法选择适当的培养液。
最全细胞培养操作手册PDF版
1/cellculture1.0 Introduction (4)2.0 Design and Equipment for theCell Culture Laboratory (4)2.1 LaboratoryD esign (4)2.2 Microbiological Safety Cabinets (5)2.3 Centrifuges (6)2.4 Incubators (6)2.5 Work Surfaces and Flooring (6)2.6 Plasticware and Consumables (7)2.7 Care and Maintenance ofLaboratoryAreas (7)3.0 Safety Aspects of Cell Culture (7)3.1 RiskAssessment (7)3.2 Biohazards (9)3.3 GeneticallyModifi ed Organisms (9)3.4 Disinfection (10)3.5 Waste Disposal (11)4.0 Sourcing of Cell Lines (12)5.0 Cell Types & Culture Characteristics (13)5.1 Primary Cultures (13)5.2 Continuous Cultures (13)5.3 Culture Morphology (13)5.4 Phases of Cell Growth (14)5.5 In Vitro Age of a Cell Culture (15)6.0 The Cell Environment (15)6.1 Basic Constituents of Media (16)6.2 InorganicSalts (17)6.3 BufferingSystems (17)6.4 Carbohydrates (17)6.5 Amino Acids (17)6.6 Vitamins (18)6.7 Proteins and Peptides (18)6.8 Fatty Acids and Lipids (18)6.9 TraceElements (18)6.10 Preparation of Media (18)6.11 Serum (19)6.12 Guidelines for Serum Use (19)6.13 Origin of Serum (20)7.0 Cryopreservation and Storage ofCell Lines (21)7.1 Cryopreservation (21)7.2 Ultra-low Temperature Storage (22)7.3 Inventory Control (23)7.4 Safety Considerations (23)8.0 Good Cell Banking Practices (24)9.0 Quality Control Considerations (26)9.1 Introduction (26)9.2 Reagents and Materials (27)9.3 Provenance and Integrity ofCellLines (27)9.4 Avoidance of Microbial Contamination (27)9.5 Environmental Monitoring (29)9.6 Aseptic Technique andContamination Control (29)9.7 What to do in the Eventof Contamination (30)10.0 Authentication of Cell Lines (31)11.0 Alternative Cell Culture Systems (31)11.1 Cell Culture Scale-up Systems (31)11.2 Scale-upSolutions (32)11.3 Roller Bottle Culture (32)11.4 MultilayerVessels (32)11.5 Disposable Solutions for Suspension Cells (34)11.6 Spinner Flask Culture (34)11.7 Other Scale-up Options (34)生命科学论坛 2ContentsCell Lines Available from ECACC (35)12.0Cell Culture Protocols ............................3612.1Basic Do’s and Don’ts of Cell Culture ........36 12.2 Protocol 1 – Aseptic Technique and GoodCell Culture Practice (37)12.3 Protocol 2 – Resuscitation of FrozenCell Lines (39)12.4 Protocol 3 – Subculture of Adherent Cell Lines (42)12.5 Protocol 4 – Subculture of Semi-AdherentCell Lines (45)12.6 Protocol 5 – Subcultureof Suspension Cell Lines (47)12.7 Protocol 6 – Cell Quantifi cation (49)12.8 Protocol 7 – Cryopreservationof Cell Lines (52)12.9 Protocol 8 – Testing for Bacteriaand Fungi (54)12.10 Protocol 9 – Detection of Mycoplasma byCulture (56)12.11 Protocol 10 – Testing for Mycoplasma byIndirect DNA Stain (58)TablesTable 1 Commonly used cell lines of eachculture type (14)Table 2 Different types of culture medium andtheir uses (16)Table 3 Comparison of ultra-low temperaturestorage methods for cell lines (22)Table 4 “Half-Way House” Solutions toScale-up (33)Table 5Cell Culture Reagents availablefrom Sigma-Aldrich .......................65Figures Figure 1 Diagram of Microbiological Safety Cabinet Airfl ow Patterns .....................5Figure 2 Examples of Cell Morphology ...........13Figure 3 Schematic Representation of a Tiered Cell Banking System .........................25Figure 4 Bioreactor ........................................31Figure 5 Hyperfl ask & T Flask ..........................31Figure 6 Shake Flasks .....................................31Figure 7 Roller Deck .......................................32Figure 8 Roller Bottles .....................................32Figure 9 Spinner Flasks ...................................32Figure 10 Flow Scheme for Bacteria and Fungi Testing ..............................................54Figure 11 Flow Scheme for Detection of Mycoplasma by Culture ....................56Figure 12 Typical ‘fried egg’ colonies, Mycoplasma pneumoniae ......................................57Figure 13 Flow Scheme for Detection of Mycoplasma by Indirect DNA Stain ....58Figure 14 Testing for Mycoplasma by Indirect DNA Stain ........................................60 (a) Hoechst Positive Culture(b) Hoechst Negative Culture 生命科学论坛 The European Collection of Cell Cultures (ECACC) was established in 1984 as a cell culture collection to service the research community and provide an International D epository Authority recognised patent depository for Europe. Over the last three decades ECACC has expanded and diversifi ed to become one of the premier collections of authenticated cell cultures in the world and this remains the core of ECACC’s business. The collections currently hold over 40,000 cell lines representing 45 different species, 50 tissue types, 300 HLA types, 450 monoclonal antibodies and at least 800 genetic disorders.The development and maintenance of such a diverse collection has inevitably produced a high level of specialist knowledge and this, combined with the support of the Health Protection Agency, has enabled ECACC to position itself as a centre of expertise in all aspects of cell culture. ECACC has developed a comprehensive range of cell culture services and diversifi ed into new product areas such as high quality genomic DNA extracted from cell lines.ECACC is one of the four collections which constitute the HealthProtection Agency Culture Collections (HPA Culture Collections).A sAs has been the case since its inception, ECACC continues tooperate out of the Porton Down site, which is now the Centre forEmergency Preparedness and Response (CEPR), Health ProtectionAgency, UK.*See page 35 for more information on the cell lines available.Culture Collectionsemail: hpacultures@rg..u k The European Collection of Cell Cultures (ECACC)Visit for more information!l Cell Lines and Hybridomas l Primary Cells l Neuron Culture Kits l HepaRG Cells l DNA & RNA Products l Bacteria, Plasmids, Transposons l Mycoplasmas l Fungil LENTICULE Discs l Viruses Cell Culture Management Services Contract Cell Culture Assay Ready Cells Cell Line Identity Verifi cation DNA Extraction Genetic Support Services Contract Freeze-Drying ProductsServices www.hp p a c u l .u ne s a nd Hybrid omasy C y n C G & R ia ia, pl a s C UL s l Ce S e l Co l As l Ce l DN l Ge l Co C ell ells sCu u lt ltu re Ki Kit s® C C e Ce lls sl NA A N Pr r odu o cts tsct P l as s m a d ds id s T , T ran ranspo spo o son s on sm sm as sLE E D D i scs cs sProducts and Services available from the HPA Culture Collections:e informa t i o n!e nt a ti es t o n l Mycoplasma Testing l Virus Contract Services l Patent Deposits l Safe Deposits l Sterility Testing l Training生命科学论坛 Introduction 4 1.0 IntroductionOver ten years ago, Sigma ® Life Science and the European Collection ofCell Cultures (ECACC) formed a working partnership to bring togetherthe most diverse selection of cell culture products and services availablecommercially. We did this with researchers like you in mind, to ensure thatyou have the necessary quality products to further your research goals.We continue to expand upon this partnership, and now are able to offeran even greater array of cell lines, cell culture products, knowledge, andservices to the global research community.The fi eld of cell culture has advanced greatly over the years. For morethan 25 years, Sigma and ECACC have both been part of and contributedto that advancement. Early cell culture research focused on discoveringmethods for culturing a diverse array of cells from many species. Today cellculture methods are vital to broad areas of life science research. With thenumber of researchers adding cell culture to their repertoire of techniquesexpanding daily, we believe there are many who can benefi t from Sigma’sand ECACC’s combined knowledge and experience in cell culture.To that aim, we have assembled this updated laboratory handbook of cellculture techniques. For the researcher new to cell culture, this handbookprovides a wealth of information from the sourcing of cell lines, safetyand laboratory design to aspects of cryopreservation and quality control.Additionally, a series of 10 detailed protocols are provided, which areroutinely used in the ECACC laboratories. For the “expert” cell culturist, itaddresses a number of important, yet often overlooked topics in cell culturesuch as cell line authentication and contamination issues, to help ensurethat the results obtained from cell culture experiments are both accurateand reproducible. The handbook is intended as a guide rather than an in-depth text book of cell culture and you are encouraged to consult relevantspecialised literature to obtain more detailed information.2.0 Design and Equipment for the CellCulture Laboratory2.1 Laboratory DesignPerhaps one of the most under-rated aspects of tissue culture is the needto design the facility to ensure that good quality material is produced in asafe and effi cient manner. Most tissue culture is undertaken in laboratoriesthat have been adapted for the purpose and in conditions that are notideal. However, as long as a few basic guidelines are adopted this shouldnot compromise the work.There are several aspects to the design of good tissue culture facilities.Ideally work should be conducted in a single use facility which, if at allpossible, should be separated into an area reserved for handling newlyreceived material (quarantine area) and an area for material which is 生命科学论坛 /cellculture5 known to be free of contaminants (main tissue culture facility). If this is notpossible work should be separated by time with all manipulations on cleanmaterial being completed prior to manipulations involving the ‘quarantinematerial’. Different incubators should also be designated. In addition, thework surfaces should be thoroughly cleaned between activities.All new material should be handled as ‘quarantine material’ until it hasbeen shown to be free of contaminants such as bacteria, fungi andparticularly mycoplasma. Conducting tissue culture in a shared facilityrequires considerable planning and it is essential that a good technique isused throughout to minimise the risk of contamination occurring.For most cell lines the laboratory should be designated to at leastCategory 2 based on the Advisory Committee on D angerous Pathogens(ACDP) guidelines (ACDP, 1995)†. However, the precise category requiredis dependent upon the cell line and nature of the work proposed. Theguidelines make recommendations regarding the laboratory environmentincluding lighting, heating, the type of work surfaces and fl ooring andprovision of hand washing facilities. In addition, it is recommended thatlaboratories should be run at air pressures that are negative to corridors tocontain any risks within the laboratory.† Advisory Committee on Dangerous Pathogens (1995) Categorisation ofBiological Agents According to Hazard and Categories of Containment,4th edition, Health & Safety Executive (HSE) books, Sudbury, UK,().2.2 Microbiological Safety CabinetsA microbiological safety cabinet is probably the most important piece ofequipment for cell culture since, when operated correctly, it will provide aclean working environment for the product, whilst protecting the operatorfrom aerosols. In these cabinets operator and/or product protection isprovided through the use of HEPA (high effi ciency particulate air) fi lters.The level of containment provided varies according to the class of cabinetused. Cabinets may be ducted to atmosphere or re-circulated through asecond HEPA fi lter before passing to atmosphere see fi gure 1.Class I Class 2Class 3known to be free of contaminants(main tissue culture facility)If this is notFigure 1. Diagram ofmicrobiological safetycabinet airfl ow patterns 生命科学论坛 6Environmental monitoring with Tryptose Soya Broth agar settle plates insidethe cabinet for a minimum of four hours is a good indicator of how cleana cabinet is (refer to ‘9.5 Environmental Monitoring’). There should be nogrowth of bacteria or fungi on such plates.In most cases a class 2 cabinet is adequate for animal cell culture.However, each study must be assessed for its hazard risk and it is possiblethat additional factors, such as a known virus infection or an uncertainprovenance may require a higher level of containment.2.3 CentrifugesCentrifuges are used routinely in tissue culture as part of the subcultureroutine for most cell lines and for the preparation of cells for cryopreservation.By their very nature centrifuges produce aerosols and thus it is necessaryto minimise this risk. This can be achieved by purchasing models that havesealed buckets. Ideally, the centrifuge should have a clear lid so that thecondition of the load can be observed without opening the lid. This willreduce the risk of the operator being exposed to hazardous material if acentrifuge tube has broken during centrifugation. Care should always betaken not to over-fi ll the tubes and to balance them carefully. These simplesteps will reduce the risk of aerosols being generated. The centrifuge shouldbe situated where it can be easily accessed for cleaning and maintenance.Centrifuges should be checked frequently for signs of corrosion.A small bench-top centrifuge with controlled braking is suffi cient for mostpurposes. Cells sediment satisfactorily at 80 – 150 x g . Higher gravitationalforces may cause damage and promote agglutination of the cell pellet.2.4 IncubatorsCell cultures require a strictly controlled environment in which to grow.Specialist incubators are used routinely to provide the correct growthconditions, such as temperature, degree of humidity and CO 2 levels in a controlled and stable manner. Generally, they can be set to runat temperatures in the range of 28o C (for insect cell lines) to 37o C (formammalian cell lines) and set to provide CO 2 at the required level (e.g. 5-10%). Some incubators also have the facility to control the O 2 levels. Copper-coated incubators are also now available. These are reported toreduce the risk of microbial contamination within the incubator due to themicrobial inhibitory activity of copper. The inclusion of a bactericidal agentin the incubator water trays will also reduce the risk of bacterial and fungalgrowth. However, there is no substitute for regular cleaning.2.5 Work Surfaces and FlooringIn order to maintain a clean working environment the laboratory surfacesincluding bench-tops, walls and fl ooring should be smooth and easyto clean. They should also be waterproof and resistant to a variety ofchemicals (such as acids, alkalis, solvents and disinfectants). In areas usedfor the storage of materials in liquid nitrogen, the fl oors should be resistantto cracking if any liquid nitrogen is spilt. Refer to Section 7.4 for safety 生命科学论坛 /cellculture7 considerations on the use of liquid nitrogen. In addition, the fl oors andwalls should be continuous with a coved skirting area to make cleaningeasier and reduce the potential for dust to accumulate. Windows shouldbe sealed. Work surfaces should be positioned at a comfortable workingheight.2.6 Plasticware and ConsumablesAlmost every type of cell culture vessel, together with support consumablessuch as tubes and pipettes, are commercially available as single use, sterilepacks. Suppliers include Sigma-Aldrich, Nunc, Greiner, Bibby Sterilin andCorning. The use of such plasticware is more cost effective than recyclingglassware, enables a higher level of quality assurance and removes theneed for validation of cleaning and sterilisation procedures. Plastic tissueculture fl asks are usually treated to provide a hydrophilic surface to facilitateattachment of anchorage dependent cells.2.7 Care and Maintenance of Laboratory AreasIn order to maintain a clean and safe working environment tidiness andcleanliness are key. All spills should be dealt with immediately. Routinecleaning should be undertaken involving the cleaning of all work surfacesboth inside and outside of the microbiological safety cabinet, the fl oors andall other pieces of equipment e.g. centrifuges. Humidifi ed incubators area particular area for concern due to the potential for fungal and bacterialgrowth in the water trays. This will create a contamination risk that canonly be avoided by regular cleaning of the incubator. All major piecesof equipment should be regularly maintained and serviced by qualifi edengineers, for example:• Microbiological safety cabinets should be checked every six monthsto ensure that they are safe to use in terms of product and userprotection. These tests confi rm that the airfl ow is correct and that theHEPA fi lters are functioning properly.• The temperature of an incubator should be regularly checked with aNAMAS (National Accreditation of Measurement and Sampling, UK),or equivalent calibrated thermometer and temperature adjusted asnecessary.• Incubator CO2and O2levels should also be regularly checked to ensurethe levels are being maintained correctly.3.0 Safety Aspects of Cell Culture3.1 Risk AssessmentThe main aim of risk assessment is to prevent injury, protect propertyand avoid harm to individuals and the environment. In many countriesthe performance of risk assessment is a legal requirement. For example,this is the case in the UK under the Health and Safety at Work Act, UKconsiderations on the use of liquid nitrogen In addition thefloors andT fl asks available fromCorning 生命科学论坛 8(1974). There are also European Community directives covering Health andSafety at work. You can visit the European Agency for Safety and Healthat Work website (www.europe.osha.eu.int) for information on legislationand standards or you should contact your on-site Health and Safetyrepresentative. Consequently risk assessments must be undertaken prior tostarting any activity. The assessment consists of two elements:1. Identifying and evaluating the risks.2. Defi ning ways of avoiding or minimising the risk.For animal cell culture the level of risk is dependent upon the cell line tobe used and is based on whether the cell line is likely to cause harm tohumans. The different classifi cations are given below:Low risk - Non human/non primate continuous celllines and some well characterised human continuouslines.Medium risk- Poorly characterised mammalian cell lines.High risk- Primary cells derived from human/primate tissue or blood.- Cell lines with endogenous pathogens (the precisecategorisation is dependent upon the pathogen) – referto ACDP guidelines, for details †.- Cell lines used following experimental infection wherethe categorisation is dependent upon the infectingagent – refer to ACDP guidelines, for details.†Advisory C ommittee o n D angerous P athogens (ACD P) (1995) C ategorisationof Biological Agents According to Hazard and Categories of Containment,4th edition, HSE books, Sudbury, UK. The second supplement to the 1995document was produced in 2000 - ‘Second supplement to: Categorisationof biological agents according to hazard and categories of containment(Fourth edition, 1995) Second Edition 2000’. Crown copyright 2000, UK.An update to the Approved List of Biological agents was issued in 2004,available at: /pubns/misc208.pdfNote: The U.S. D epartment of Health and Human Services (Centers forDisease Control and Prevention) publishes a similar list, in its Biosafety inMicrobiological and Biomedical Laboratories (BMBL) document (2007).The U.S. system uses Biological Safety Levels in place of the UK ACD Phazard groups.A culture collection such as ECACC will recommend a minimumcontainment level required for a given cell line based upon its riskassessment. For most cell lines the appropriate level of containment isLevel 2 requiring a class 2 microbiological safety cabinet. However, thismay need to be increased to containment Level 3 depending upon the(1974). There are also European Community dir 生命科学论坛 /cellculture9 type of manipulations to be carried out and whether large culture volumesare envisaged. For cell lines derived from patients with HIV or HumanT-Lymphotropic Virus (HTLV) Level 3 containment is required.Containment is the most obvious means of reducing risk. Other less obviousmeasures include restricting the movement of staff and equipment into andout of laboratories. Good laboratory practice and good bench techniquessuch as ensuring work areas are uncluttered, reagents are correctly labelledand stored, are also important for reducing risk and making the laboratorya safe environment in which to work. The risk of exposure to aerosols orsplashes can be limited by avoiding rapid pipetting, scraping and pouring.In addition, it is recommended that people working in laboratories whereprimary human material is used are vaccinated against Hepatitis B. Stafftraining and the use of written standard operating procedures and riskassessments will also reduce the potential for harm. Cell culture trainingcourses covering the basics of tissue culture safety are offered by ECACC.3.2 BiohazardsViruses pathogenic for humans are one of the most likely biohazardspresented by cell cultures. Where infection with an agent pathogenic forhumans is known or suspected, the cell culture should be handled at acontainment level appropriate for the agent concerned. Other potentialbiohazards should also be considered. These relate to components ofthe cell culture medium, other adventitious agents (e.g. contaminatingmycoplasmas), and cell products, some of which may be biologicallyactive molecules with pharmacological, immunomodulating or sensitisingproperties. In addition, the generation and use of modified cells, forexample, hybrids, transformed cells and cells containing recombinantD NA can be hazardous. These procedures could potentially result in theappearance of modifi ed or reactivated viruses, novel fusion/hybrid proteins(especially in cross-species hybrids) and the expression of viral or cellularoncogenes.Laboratory workers should never culture their own cells. In vitrotransformation or genetic modification could result in malignant diseaseor expression of an unusual pharmacologically active protein if they wereto be accidentally inoculated into the donor. Therefore, human cells shouldbe obtained from individuals having no association with the experimentalwork.Biohazardous waste should be disposed of according to the methodsdescribed under ‘3.5 Waste Disposal’.3.3 Genetically Modifi ed OrganismsThe generation and use of genetically modifi ed organisms (GMOs) shouldbe strictly controlled and regulated. Most countries have regulatoryorganisations to ensure the risks posed by GMOs are minimised. Forexample, in the UK all institutions that carry out work using and/orgenerating GMOs are required by law to have a Genetic Modifi cationtype of manipulations to be carried out and whether large culture volumes生命科学论坛 10Safety Committee (GMSC). Prior to any work commencing proposals forthe intended work should go through the committee and , if necessary, beapproved by the Health and Safety Executive (HSE). There is a EuropeanDirective governing the regulation of GM work. Visit the European Agencyfor Safety and Health at Work website (www.europe.osha.eu.int) forinformation on legislation and standards, or contact your on-site Healthand Safety representative.It is the responsibility of the individual cell culture user and institutionto ensure compliance with the regulations set by the authorities of thecountry they are operating in.3.4 DisinfectionMethods designed for the disinfection/decontamination of culture waste,work surfaces and equipment represent important means for minimisingthe risk of harm. Always wear appropriate personal protective equipment(PPE) such as gloves and eye protection when using concentrated formsof disinfectants. The selected gloves should protect against the substancebeing handled and meet the European standard EN374-3. Manufacturers’’charts will help to identify the best gloves for the work.The major disinfectants fall into four groups and their relative merits canbe summarised as follows:Hypochlorites (e.g., Sodium Hypochlorite)• Good general purpose disinfectant• Active against viruses• Corrosive against metals and therefore should notbe used on metal surfaces e.g. centrifuges• Readily inactivated by organic matter and thereforeshould be made fresh daily• Should be used at 1000ppm for general use surfacedisinfection, 2500ppm in discard waste pots fordisinfecting pipettes, and 10,000ppm for tissue culturewaste and spillages Note: When fumigating a cabinet or room using formaldehyde all thehypochlorites must fi rst be removed as the two chemicals react together toproduce carcinogenic products.PhenolicsPhenolic based disinfectants should never be used as they are notsupported as part of the EU Biocidal Products Directive reviewprogramme.Alcohol (e.g. Ethanol, Isopropanol)• Effective concentrations: 70% for ethanol, 60-70%for isopropanol • Their mode of activity is by dehydration and fi xation Safety Committee (GMSC). Prior to any work 生命科学论坛 /cellculture11• Effective against bacteria. Ethanol is effectiveagainst most viruses but not non-enveloped viruses• Isopropanol is not effective against virusesAldehydes (e.g. Formaldehyde)• Aldehydes are irritants and their use should belimited due to problems of sensitisation• Should only be used in well ventilated areas.Formaldehyde is used to fumigate laboratories. The formaldehye isheated in a device so it will vaporise and all exposed surfaces arecoated with the disinfectant.Generally the use of aldehydes for disinfection and fumigationpurposes can be hazardous. Check local regulations and with yoursafety advisor.3.5 Waste DisposalAny employer has a ‘duty of care’ to dispose of all biological waste safelyin accordance with national legislative requirements. Given below is a listof ways in which tissue culture waste can be decontaminated and disposedof safely. One of the most important aspects of the management of alllaboratory-generated waste is to dispose of waste regularly and not toallow the amounts to build up. The best approach is ‘little and often’.Different forms of waste require different treatment.• Tissue culture waste (culture medium) – inactivate for at least 2 hoursin a solution of hypochlorite (10,000ppm) prior to disposal to drainwith an excess of water.• Contaminated pipettes should be placed in hypochlorite solution(2500ppm) overnight before d isposal by autoclaving and incineration.• Solid waste such as fl asks, centrifuge tubes, contaminated gloves,tissues, etc., should be placed inside heavy-duty sacks for contaminatedwaste and incinerated.• If at all possible waste should be incinerated rather than autoclaved.• Waste from specially licensed laboratories e.g. those handlinggenetically modifi ed level 3 (GM3) organisms requires specifi ctreatment and tracking.•Effective against bacteria Ethanol is effectiveSafety Aspects of Cell Culture生命科学论坛 。
动物细胞培养技术实验指导(全面)
实验篇实验一实验器材的清洗实验物品（一）每一大组公用物品（每班分6大组）吸球4个、酒精棉球瓶两个、消毒水一瓶、吸瓶两个、计数板1块、洗液缸一个、95%酒精1瓶。
（二）每一小组公用物品（两人一小组）刻度吸管5毫升4支, 小漏斗1个、 80-200铜滤网1块、培养皿2个、 l00毫升盐水瓶及塞子4个、l0毫升盐水瓶及塞子1个、100ml培养瓶及塞子4个、10ml 培养瓶及塞子1个、镊子1个、剪刀1把、冻存管（1.5毫升，2毫升）2个、软毛刷2个、塑料盆一个。
（二）公用物品小滤器、大滤器2个、1000毫升量筒2个、100毫升量筒2个、家用剪刀、耐酸乳胶手套长的两双、短的四双、酒精一壶、玻璃棒几个、 0.22μm的小滤膜一盒、电炉3个。
清洗注意事项和要求（一）使用后的实验器材应立即投入清水中。
带毒的玻璃器皿需先浸泡在5％来苏儿或1％盐酸溶液中（依病毒的种类而定）1天以上或高压灭菌。
（二）浸泡、煮沸、酸泡的器皿内要充满液体，不得有气泡。
（三）煮沸前的水面要高于器材5厘米，水沸后投入洗涤剂（直径35厘米的铝锅，用洗衣粉10克左右）；若洗涤剂和实验器材同时从冷水煮至沸腾或使用过量洗剂均易腐蚀玻璃表面，使玻璃碱化PH值上升。
（四）软毛刷的刷端已掉毛的应该弃去，否则会损害玻璃。
玻璃划痕处易残留洗涤剂,会改变培养液PH和毒害细胞。
（五）浸泡器材的蒸馏水容器要专用，并做好标记，如“蒸馏水1盆”、”蒸馏水2盆。
（六）器材清洗干燥后，在以后各步操作时，手指不可接触器材的使用端。
清洗者可戴一次性薄膜手套进行操作，这样省时又保证清洗质量。
（七）清洁物品应及时包装消毒，应注意妥善保存，防止落人灰尘、蟑螂、蚂蚁等引起二次污染。
（八）刷洗、酸泡后的器材要用流水振荡冲洗；不得残留洗涤剂、清洁液。
操作方法（一）清洗液（俗称酸液）的配制方法常用清洁液配方重铬酸钾100g，浓硫酸200ml，蒸馏水800ml。
以配置10000ml常用清洁液为例介绍如下：先在搪瓷盆或塑料盆中加入蒸馏水8000ml，称取1000g重铬酸钾，用玻璃棒搅拌直至溶解（可加热以辅助溶解），待重铬酸钾液冷却后，缓慢加入浓硫酸，边加边用玻璃棒搅动，以混合液温度不过快上升和不出现重铬酸钾结晶为度。
细胞培养操作指南
细胞培养操作指南1.实验室准备：在开始细胞培养之前，准备好所有所需的实验室设备和试剂：培养皿、细胞培养基、胎牛血清、无菌移液器、无菌离心管、显微镜等。
2.个人准备：进入实验室前，务必穿戴好个人防护装备，包括实验室服、手套和口罩。
确保双手清洁，并使用含酒精的消毒液消毒工作台。
3.细胞培养基准备：根据实验需求，配制适当的细胞培养基。
将细胞培养基加热至37摄氏度，使其变得温暖。
4.细胞扩增与传代：a.从冷冻保存的细胞存储管中取出细胞，并将其转移到一只预先加热的离心管中。
b.离心管轻轻离心，使细胞沉淀在管底。
c.弃去上清液，加入预先加热的新鲜培养基，轻轻悬浮细胞沉淀。
d.将细胞悬液转移到一个新的培养皿中，确保细胞均匀分布。
e.将培养皿放入恒温培养箱，并根据细胞类型和实验要求设置合适的温度和湿度。
f.观察细胞生长情况，并根据需要传代细胞。
传代细胞时，将细胞悬液转移到新的培养皿中，注意细胞定植的密度不要太高。
5.细胞分裂与凝聚度检测：使用显微镜观察细胞培养的生长情况，检查细胞的形态和凝聚度。
正常细胞应该呈现光滑、扁平、并且凝聚在一起的形态。
6.细胞的移植和收获：当细胞生长到适当的数量时，可以进行细胞的移植或收获。
使用无菌移液器将细胞转移至其他试管或培养皿中，确保操作环境和使用器皿都是无菌的。
7.细胞冻存：根据实验的需要，将细胞制备成冻存物。
将细胞悬液转移到冻存保存培养基中，并加入适量的冷冻保护剂（如DMSO），在-80摄氏度的冰箱中快速冷冻。
8.废弃物处置：在培养过程中产生的废弃物（例如培养皿、试管等），必须按照实验室的规定进行处理。
通常情况下，将废弃物放入带有生物危险标识的废物箱中。
9.清洗和消毒：在培养工作完成后，清洗并消毒实验室工作台和使用的器皿。
使用含酒精的消毒液对工作台表面进行擦拭，并将使用过的器皿彻底清洗，并经过高温高压灭菌。
10.实验记录：进行细胞培养时，要详细记录每个操作步骤，包括使用的细胞类型、培养方法、检测结果等。
《细胞生物学》实验指导书(不太全供参考).doc
《细胞生物学》实验指导书（不太全供参考）适用专业:生物科学、生物技术实验目录实验一植物原生质体的分离与融合•••]实验二动物细胞原代培养•••3实验三细胞传代培养…彳实验四植物细胞骨架的光学显微镜观察••Y实验五细胞凋亡的诱导、观察与检测“I实验六环境因素诱变染色体改组的观察实验七红细胞膜蛋白的分离及其电泳检测 (11)实验一植物原生质体的分离与融合实验项目类型:综合性所属课程名称：《细胞生物学》实验计划学时：7学时一、实验目的1.掌握原生质体分离的原理与技术；2.了解细胞融合的基本原理及应用；3.初步掌握PEG融合法和电融合法.二、实验原理植物原生质体是去除了细胞壁的裸露的细胞.原生质体可以从培养的单细胞、愈伤组织和植物器官（叶等）获得.但一般认为从叶肉组织分离原生质体是理想的材料，其优点是材料来源方便，供应及时，而且遗传性较为一致.原生质体的分离通常采用酶解法，对细胞壁成分进行降解后获得.原生质体培养的条件和对营养的要求与组织、细胞相似.但原生质体由于除去了细胞壁,所以需要一定浓度的渗透压稳定剂来保持原生质体的稳定.常用的渗透压稳定剂包括甘露醇、山梨醇、蔗糖等. 其次，还应当考虑取材、酶的种类和纯度、酶液的渗透压、酶解时间及温度等因素对分离原生质体的影响.2个或2个以上的细胞合并成为1个细胞的现象称为细胞融合.细胞融合的主要方法有病毒法、聚乙二醇(PEG)法和电融合3种方法，其原理基本相同，都是两细胞接触点的膜分子发生重组,然后由于表面张力作用,形成一个球形细胞. 原生质体分离融合和培养的意义：1.除去了细胞壁为植物细胞之间的融合扫平了障碍，为制造新杂种开辟了道路.植物原生质体融合和培养在植物遗传工程和育种研究上具有广阔的应用前景.它是植物同源、异源多倍体获得的途径之一，它不仅能克服远缘杂交有性不亲和障碍，也可克服传统的通过有性杂交诱导多倍体植株的麻烦，最终将野生种的远缘基因导入栽培种中，原生质体融合技术可望成为作物改良的有力工具之一.2.原生质体可摄入外源DNA,细胞器、细菌或病毒颗粒,这些特性与植物全能性相结合为高等植物的遗传饰变打下基础.3.获得细胞无性系和选育突变体的优良起始材料.三、实验仪器与材料1.材料:油菜叶子、白菜花2.溶液或试剂:(1)洗涤液:甘露醇0.7mol/L,CaC12 • 2H2O3.5mmol/L,KH2PO4 0.7mmol/L(pH 5.6),高压灭菌；(2)混合酶液:1.5%纤维素酶,1%果胶酶溶于洗涤液中(pH 5.6).(3)50%PEG(4)高pH高钙稀释液：(5)20%蔗糖溶液(6)DPD培养基：3.仪器或其他用具:超净工作台、灭菌锅、显微镜、恒温培养箱、镶子、解剖刀、剪子、接种针、铝饭盒、锡箔纸、记号笔、橡皮筋、试剂瓶、三角瓶移液管、培养皿、酒精灯等.四、实验步骤(一)原生质体的制备：1.取材:取新鲜油菜叶和白菜花瓣自来水洗净，再用70%乙醇浸泡30S,0.5%次氯酸钠消毒lOmin,无菌水冲洗5次.2.酶解：材料切成1mm宽细丝，加入适量酶液，置摇床上(60〜70rpm),在25〜28°C黑暗条件下，酶解5〜7h.3.分离：用200目网过滤除去未完全消化的残渣，在lOOOrpm条件下离心5分钟,弃上清.加入3〜4ml洗涤液，相同条件下离心2-5分钟，弃上清，留1ml洗液.用滴管将混有原生质体的1ml洗液吸出，轻轻铺于20% 蔗糖溶液上(5ml离心管装3ml 20%蔗糖溶液)，在lOOOrpm条件下离心5-10分钟，由于密度梯度离心的作用，生活力强状态好的原生质体漂浮在20%的蔗糖与洗涤液之间，破碎的细胞残渣沉入管底.用200 u 1移液器轻轻将状态好的原生质体吸出（注意尽可能不要吸入下层的蔗糖溶液），放入另一干净的离心管中，加4ml洗涤液,1000rpm离心2-5分钟, 弃上清.（二）原生质体融合：1.将原生质体用DPD调节浓度为1*105个/mL.2.将原生质体混合物滴入小培养皿，静置8〜10分钟.3.然后滴入50%的PEG溶液，静置2分钟.4.依次间隔5分钟加入0.5ml、1 ml和2 ml高钙高pH洗涤液.注意在第二、三次洗液加入前,用移液器轻轻吸走部分溶液，但不能吸干，否则原生质体破碎死亡;最后用液体培养基洗1〜2次.制片，镜检.五、实验报告画出观察到的融合细胞，并计算融合率.六、思考题1.你认为要获得数量多、生活力强的原生质体，在实验中应注意那些问题？增多数量的方法:多取材且尽量切碎，增加酶浓度，提高酶解温度，延长酶解时间，操作轻柔，勿使原生质体破碎等.提高生活力的方法:取新鲜材料,采用合适的酶浓度、作用温度和时间2.举例说明原生质体融合的应用价值.实验二动物细胞原代培养实验项目类型:基础性所属课程名称：《细胞生物学》实验计划学时：4学时一、实验目的1.掌握动物细胞原代培养的基本方法和操作过程；2.熟悉原代培养细胞观察方法.二、实验原理用直接从机体获取的细胞进行的培养称为原代培养.原代培养是建立各种细胞系的第一步，该技术可以在体外进行各种类型细胞的增殖、遗传、变异、分化和脱分化、恶变与去恶变等研究.分为组织块培养法和消化法两种.三、实验仪器与材料1.材料:小鼠心、肝、脾、肾等2.溶液或试剂：(1)RPMI-1640培养基、小牛血清.(2)0.25%胰蛋白酶、(pH 5.6).(3)Hanks液3.仪器或其他用具:CO2培养箱、倒置显微镜、超净工作台、高压锅、水浴锅、解剖器械、培养瓶、微量加样器、吸管、移液管、酒精灯、酒精棉球等.四、实验步骤1.处死小鼠:将小鼠用颈椎脱臼法处死，放入75%酒精种浸泡消毒.2.取材:在超净工作台中用灭菌的解剖器械剖开小鼠腹腔，辨认并取下肝脏、脾脏和肾脏，放入灭菌的培养皿中，加入Hanks液清洗，然后加入2mL胰蛋白酶液,研磨并过滤.3.将液体转移到离心管,1200rpm离心5min收集细胞,加入适量培养液,C02培养箱培养.4.观察:逐日在倒置镜下观察细胞生长情况.五、实验报告画出细胞贴壁前和贴壁后的形态.六、思考题试述细胞原代培养成功的条件？实验三细胞传代培养实验项目类型:基础性所属课程名称：《细胞生物学》实验计划学时：3学时一、实验目的1.熟练掌握贴壁细胞传代的培养方法；2.观察传代细胞贴壁、生长、繁殖过程中细胞形态的变化.二、实验原理离体培养的细胞群体增殖达到一定密度时，细胞的生长和分裂速度就会减慢甚至停止，如不及时分离传代培养,细胞将逐渐衰老死亡.传代培养是指细胞从一个培养瓶以1:2或其它比率转移，接种到另一培养瓶的培养.贴壁培养细胞的传代通常采用胰蛋白酶消化,把细胞分散成单细胞再传代.三、实验仪器与材料1.材料:原代培养的小鼠细胞或Hela细胞2.溶液或试剂：(1)RPMI-1640培养基、小牛血清.(2)0.25%胰蛋白酶、(pH 5.6).(3)Hanks液3.仪器或其他用具:CO2培养箱、倒置显微镜、超净工作台、高压锅、水浴锅、培养瓶、微量加样器、吸管、移液管、酒精灯、酒精棉球等.四、实验步骤1.将长成单层的细胞从二氧化碳培养箱中取出,在超净工作台中倒掉瓶内的培养液，加入少许消化液.(以液面盖住细胞为宜)，静置5~10分钟.2.在倒置镜下观察被消化的细胞，如果细胞变圆,相互之间不再连接成片，这时应立即在超净台中将消化液倒掉，加入3〜5ml新鲜培养液,吹打, 制成细胞悬液.3.将细胞悬液吸出2ml左右，加到另一个培养瓶中并向每个瓶中分别加3ml 左右培养液，盖好瓶塞，送回二氧化碳培养箱中，继续进行培养.五、实验报告分别画出贴壁生长的小鼠细胞和Hela细胞,并说明二者的不同.六、思考题写出细胞传代培养成功的条件？实验四植物细胞骨架的光学显微镜观察实验项目类型:基础性所属课程名称：《细胞生物学》实验计划学时：3学时一、实验目的了解细胞骨架的结构特征及其制备技术二、实验原理细胞骨架是由蛋白质丝组成的复杂网状结构，根据其组成成分和形态结构可分为微管、微丝和中间纤维.它们对细胞形态的维持，细胞的生长、运动、分裂、分化，物质运输，能量转换，信息传递，基因表达等起到重要作用.用适当浓度的Triton X-100处理细胞时，可将细胞质膜和细胞质中的蛋白质和全部脂质溶解抽提，但细胞骨架系统的蛋白质不受破坏而被保存，经戊二醛固定，考马斯亮蓝R250染色或间接免疫荧光标记以后，可在显微镜下观察到细胞骨架结构.三、实验仪器与材料1.材料:洋葱鳞茎和口腔上皮细胞2.溶液或试剂：(1)M-缓冲液(2)6mmol/L(pH 6.8)磷酸缓冲液(3)l%Triton X-100(用M-缓冲液配制)(4)0.2%考马斯亮蓝R250(5)3%戊二醛3.仪器或其他用具:光学显微镜、50ml烧杯、玻璃滴管、容量瓶、试剂瓶、载玻片、盖玻片、镶子、小剪刀、吸水纸、擦镜纸等.四、实验步骤1.撕取洋葱鳞叶内表皮若干片,大小约lcm2,置于青霉素小瓶或小烧杯中.2.用磷酸缓冲液(PBS)浸泡片刻.3.吸去磷酸缓冲液，用1% TritonX-100处理20分钟抽提细胞骨架以外的蛋白质,从而使骨架图像更加清晰.4.吸去TritonX-100,用M-缓冲液洗3次，每次3分钟.M-缓冲液有稳定细胞骨架的作用.5.用3%戊二醛固定15-20分钟6.用PBS洗3次，每次3分钟7.考马斯亮蓝染色15分钟8.蒸馅水洗2次9.置于载玻片上，盖上盖玻片，在光学显微镜下观察.五、实验报告绘出植物细胞骨架微丝结构图.六、思考题说出各步骤的目的和一些重要试剂的在此处的作用.实验五细胞凋亡的诱导、观察与检测实验项目类型:创新设计性实验所属课程名称：《细胞生物学》实验计划学时：9学时一、实验目的1.了解细胞凋亡的概念和原理;2.掌握细胞凋亡诱导的方法;3.认识细胞凋亡的特征.二、实验原理人体内的细胞注定是要死亡的，目前人们已经知道细胞的死亡起码有两种方式，即细胞坏死与细胞凋亡.坏死是细胞受到强烈理化或生物因素作用引起细胞无序变化的死亡过程.表现为细胞胀大，胞膜破裂，细胞内容物外溢，核变化较慢,DNA降解不充分，引起局部严重的炎症反应.凋亡是细胞受到内、外因子刺激后发生的由基因调控的生理性死亡行为.其细胞及组织的变化与坏死明显不同.细胞凋亡的形态学变化: 首先细胞体积缩小，连接消失，与周围的细胞脱离，然后胞质密度增加, 核质浓缩，核膜核仁破碎,DNA降解,胞膜形成小泡，最终为为几个凋亡小体，无内容物外溢,因此不引起周围的炎症反应，凋亡小体可迅速被周围专职或非专职吞噬细胞吞噬.凋亡细胞DNA的有控降解是一种内源性核酸内切酶作用的结果,该酶在核小体连接部位切断染色体DNA,这种降解表现在琼脂糖凝胶电泳中就呈现特异的梯状Ladder图谱,而坏死呈弥漫的连续图谱.三、实验仪器与材料1.鸡血细胞2.溶液或试剂：生理盐水,20mmol/LCaC12顺伯，姬姆萨染色剂，Tris.HCl,SDS, EDTA,乙醇，甲醇，蛋白酶K.3.仪器或其他用具:超净工作台、灭菌锅、显微镜、恒温培养箱、镶子、解剖刀、剪子、接种针、铝饭盒、锡箔纸、记号笔、橡皮筋、试剂瓶、三角瓶移液管、培养皿、酒精灯等.四、实验步骤（一）细胞凋亡的诱导：1.采鸡血10ml,加0.85%的生理盐水混匀,1200r/min离心5min,弃上清液（重复三次），制备红细胞悬液，然后加入50ml血细胞保存液混匀放于4°C冰箱中备用.2.取4个试管，各加入鸡血细胞保存液2ml,在1号管中加入2ml生理盐水作为阴性对照,2号试管加入2ml 10ug/ml的顺伯溶液作为阳性对照;3号试管中加入2ml 20mmol/L的CaC12溶液进行胁迫处理;4号试管不加任何物质，实验时煮沸5 min使细胞坏死.（二）细胞凋亡的形态学观察：1.处理4h取样，用生理盐水稀释5倍，各取50ul细胞悬液均匀涂布于5 片载玻片上,晾干.2.用甲醇固定2min,然后在载玻片上滴加适量姬姆萨染液染色lOmin.3.用蒸馅水轻轻洗去染液，室温晾干，在光学显微镜下观察细胞形态变化.4.照相，并对试验组细胞进行凋亡计数统计.（三）DNA梯状条带的检测：1.离心收集鸡血细胞，弃上清.2.加入1ml的细胞裂解缓冲液重悬，转入1.5ml离心管中，加入蛋白酶K （500ug/ml）20 n 1,混匀.3.在65°C恒温水浴锅中水浴30min（也可转入37°C水浴12〜24h）,间歇振荡离心管数次.4.于台式离心机以12000 rpm离心5min,取上清液入另一离心管中.5.加2倍体积异丙醇，倒转混匀后，可以看见丝状物，用100ul吸头挑出, 或12,000转/分离心5分钟，弃上清,晾干.6.用50ulTE 缓冲液(pH8.0)溶解沉淀,并加入RNase A(10ug/ul)5ul,于37 °C 保温10〜15分钟.7.1.2 %琼脂糖凝胶,75 V电压，电泳45 min左右，8.紫外灯下观察并照相.(琼脂糖凝胶电泳:称取 1.2克琼脂糖加入100ml1*TBE),加热使之溶解，取一个凝胶模子，两端用胶布封好，水平放置.将制孔器(即梳子)放在模子一侧约0.5cm处，其底部距模子约1mm.待琼脂冷至约70°C时，加入10ul 0.5mg/ml EB,混匀，倒入模子，待冷却凝固后，取下梳子，撕去两端胶布,放入电泳槽中，使有加样孔的一端放在负极一端.在电泳槽中加入1*TBE至超过凝胶表面约1mm.加入DNA 电泳样:1.5ul DNA 。
细胞培养完整手册
细胞培养完整手册引言细胞培养是现代生物学和医学研究领域必不可少的一环。
本文将介绍细胞培养的基本原理、操作步骤、培养常见问题及解决方法。
细胞培养基本原理细胞培养是指利用人工制备的培养基以及适当的生长因子、激素和营养物质等对细胞进行体外培养和扩增，从而使细胞可以在体外连续增殖并保持原有的生物学特性。
细胞培养是现代细胞生物学、免疫学、病毒学、分子生物学以及药学研究的基础。
细胞培养的操作步骤实验前准备实验前要准备工具、试剂和消毒物品等，以确保实验的顺利进行和细胞的纯度与无菌。
细胞解冻1.从低温冻存罐中取出样品，将其放在37℃恒温水浴中解冻，不要使用冷却液或热盘等快速解冻方式。
2.用培养基缓慢悬浮细胞，宜加入10% FBS卡死细胞，避免冷冻完再活化产生过多氧化物等致细胞杀伤性的物质。
3.对细胞悬浮液进行离心，弃掉上清液，用适量的完整培养基重悬细胞沉淀，充分混匀后接续培养。
细胞传代1.用 PBS 泡洗清除细胞上附着的蛋白质和细胞碎片等杂质。
2.加入胰酶 0.05% 消化 3~5 分钟，让细胞分离迅速、均匀，切勿长时间消化。
3.将消化细胞在上清液中轻轻悬浮，检查细胞的分离和体积大小。
4.将细胞上清液转移至新的培养瓶中，鉴定细胞标记和污染情况。
5.加适量的完整培养基，并放入 CO2 培养箱中进行培养，定期观察细胞的生长情况，每两到三天定期更换培养基。
技巧提示1.培养瓶应洗涤干净，并喷壁消毒剂。
2.常用的培养基配方包括 DMEM 培养基、RPMI 1640 培养基、F12 培养基等，根据不同的实验需要进行选择。
3.培养箱应定期清洗和消毒，保证无菌环境。
4.细胞的传代次数不应过多，避免引起细胞特性的变化和突变。
细胞培养常见问题及解决方法细胞失活或死亡1.培养基中缺失营养物质如血清、氨基酸等，应加入完整培养基进行补充。
2.细胞感染，需要添加抗生素，如青霉素、链霉素等。
3.培养条件不适宜，如 CO2 浓度、温度等，应适当调节。
细胞培养实验
实验一、实验器材的清洗、包装和消毒以及实验室常用的仪器设备的使用和维护一、实验目的1、认识常用实验器材。
2、实验器材的清洗。
3、实验器材的包装和消毒。
4、了解细胞培养中常用仪器设备的使用方法、注意事项和保养方法。
二、实验用品1仪器压力蒸汽消毒器、电热干燥箱、超净台、自动双重纯水蒸馏器、CO2培养箱、电热恒温培养箱、真空抽气泵或电动吸引器、液氮罐、普通冰箱、超低温冰箱2试剂及耗材（1）试剂：盐酸或硫酸、洗涤用品、消毒剂等（2）耗材：培养瓶、盐水瓶、漏斗、量筒等玻璃器皿、培养皿等塑料品、解剖器械、铝饭盒、酒精灯和记号笔等用品三、实验步骤一）玻璃器械洗消：（一）新的玻璃器皿的洗消：1.自来水刷洗，除去灰尘。
2.烘干、泡盐酸：烤箱中烘干，然后再浸入5％稀盐酸中12小时以除去脏物、铅、砷等物。
3.刷洗、烘干：12小时后立即用自来水冲洗，再用洗涤剂刷洗，自来水冲干净后用烤箱烘干。
4.泡酸、清洗：用清洁液（重铬酸钾120g：浓硫酸200ml：蒸馏水1000ml）浸泡12小时，然后从酸缸内捞出器皿用自来水冲洗15次，最后蒸馏水冲洗3-5次和用双蒸水过3次。
5.烘干、包装：洗干净后先烘干，然后用牛皮纸（油光纸）包装。
6.高压消毒：包装好的器皿装入高压锅内盖好盖子，打开开关和安全阀，当蒸气成直线上升时，关闭安全阀，当指针指向15磅时，维持20-30分钟。
7.高压消毒后烘干（二）旧的玻璃器皿的洗消：1.刷洗、烘干：使用过的玻璃器皿可直接泡入来苏尔液或洗涤剂溶液中，泡过来苏尔溶液（洗涤剂）的器皿要用清水刷洗干净，然后烘干。
2.泡酸、清洗：烘干后泡入清洁液（酸液），12小时后从酸缸内捞出器皿立即用自来水冲洗（避免蛋白质干涸后粘附于玻璃上难以清洗），再用蒸馏水冲洗3次。
3.烘干、包装：洗干净的器皿烘干后取出用牛皮纸（油光纸）等包装，以便于消毒储存及防止灰尘和再次被污染。
4.高压消毒：包装好的器皿装入高压锅内，盖好盖子，打开开关和安全阀，随着温度的上升安全阀冒出蒸气，当蒸气成直线冒出3-5分钟后，关闭安全阀，气压表指数随之上升，当指针指向15磅时，调节电开关维持20-30分钟即可。
细胞培养实验指导
细胞培养实验指导陈轶霞刘俊林实验报告书写要求：1、学生实验后应按时完成并提交实验报告。
报告要求结构完整、内容充实、图表齐全、书面整洁、分析讨论认真合理等。
2、严禁互相抄袭实验报告，发现雷同实验报告一律视为不及格。
3、切忌全班同学统一打印相同的实验结果图（以前曾发生过全班同学共用一张错误的图的情况）实验一细胞培养实验室介绍及使用卫生要求【目的与要求】1.了解细胞培养实验室的基本设施和布局2. 掌握细胞培养实验室的使用卫生要求【基本原理】细胞培养必须无菌操作，要求工作环境和条件必须保证无微生物污染和不受其它有害因素影响，应建立专门的实验室并划分不同功能区。
理想的细胞培养实验室可分为准备室、更衣室、缓冲室、培养室等。
【内容与方法】1．通过教师讲解并参观了解开展本课程教学的细胞培养实验室的布局、设施与仪器设备的摆放情况2.使用卫生要求。
（1）实验室的消毒实验室日常采用紫外灯消毒；按实验室的卫生管理要求对实验室洁净区按周期进行熏蒸消毒；在培养期间发生严重污染时要进行熏蒸消毒。
（2）培养工作开始操作前将所有培养用的物品经专用物流通道通过传递窗放进培养室，开紫外灯消毒30min，关紫外灯后方进入操作。
配有空气净化系统的洁净室在风机开启至少30min后才能达到相应的洁净度级别。
（3）操作者进入培养室时要遵守的程序a: 用肥皂洗手，在干手器上吹掉手臂上的水滴。
b: 换穿无菌室专用鞋，进入更衣室，外衣挂到指定的衣柜里，用0.1%新洁尔灭溶液对手及前臂进行消毒，戴帽子（不露头发）、口罩（应包住胡须），穿已消毒的洁净服，进入缓冲室。
再用0.1%新洁尔灭溶液对手及前臂消毒，在其滞留3min后进入培养室。
（4）开超净工作台10min，期间用0.1%新洁尔灭溶液的擦拭操作台面后可开始具体的操作，同时点燃酒精灯用于临时性的火焰消毒。
（5）实验完毕，关闭超净工作台，及时清理实验用品，废物从通往物流通道的传递窗中传出。
工作台面用0.1%新洁尔灭溶液的擦拭消毒后方可出培养室。
细胞培养大实验ppt课件
注意：镜下偶见由两个以上细胞组成的细胞团，应按单个细胞计算，若 细胞团占10%以上，说明分散不好，需重新制备细胞悬液。
血球计数板
细胞克隆化——有限稀释法
1 显微镜观察培养细胞生长状态
2 制备细胞悬液（同贴壁细胞传代） 3 用培养液细胞细胞悬液。
4 用血球计数板准确计数细胞
5 用培养基调整细胞悬液的细胞数至1×105/mL细胞 6 用无血清培养基将细胞稀释至103/mL细胞
注意事项
细胞培养应严格执行无菌操作： 1.先用酒精棉球将手擦干净。
2.取用无菌物品如培养瓶时尽量用镊子（镊子、玻璃滴管等物品用前要在
酒精灯火焰上焰烧2-3秒左右）。 2.细胞培养瓶、离心管等拿出超净台时一定要将瓶盖拧紧，避免污染细胞 3.注意避免交叉污染，尤其是吸取培养基时，一定要用无菌干净的吸管。 4.培养基尽量放在台面的最里面，培养基瓶盖打开后要倒放。拿东西时尽
4 去上清 5 加入细胞冻存液（培养基中含10% DMSO （二甲亚砜），20-50%
小牛血清）
6 放置室温冻存盒中，封闭严实 7 置-80 ℃冰箱过夜
8 转入液氮中 （－196℃ ）
细胞的复苏
1 从液氮中取出冷冻管（如为安瓶或无螺帽冷冻管，应防突然爆裂而
伤皮肤和眼睛） 2 立即放入37℃水浴中，振荡，使之尽快融化（1～2分钟），如不是
螺旋盖冷冻管，则要用镊子夹住冷冻管，防止水进入而造成污染。
3 将细胞悬液转入10 mL 预热的培养液中，混悬，离心洗涤， 1200rpm，5min。
4
去上清，加入培养液，调细胞浓度为1～2×105 /mL
5转入细胞培养瓶中培养
卫生清洁
实验完成后请各位同学将自己组的卫生清理干净：
2013《细胞培养》实验讲义
《细胞培养》实验讲义实验一细胞培养室无菌环境的建立和实验器具的清洗、包装和灭菌一、原理：防止污染是决定细胞培养成功的首要条件。
由于体外细胞缺乏抗感染能力，因此无菌技术在细胞培养中显得尤为重要。
二、目的要求：通过实验，培养学生良好的卫生观念，确立细胞培养的无菌意识；掌握细胞培养室的消毒及灭菌方法；掌握培养用品的高温消毒方法。
通过细胞培养室和主要设备的无菌环境建立，逐步掌握无菌操作概念。
掌握细胞培养室、培养箱和超净台的消毒方法；掌握玻璃器皿、金属器械、橡胶制品的清洗、包装和灭菌方法。
三、实验材料：细胞培养室环境、培养箱、超净台、边台、紫外线灯、70％酒精等等；玻璃培养瓶、玻璃滴管、手术器械、圆筒、铝制饭盒等电动高压锅、烘干箱、超声洗涤机、酸液及酸缸、纯水仪等四、操作方法：1、环境消毒细胞培养室环境消毒用紫外线灯照射30～60分钟以上才能进入其内工作。
培养箱箱体内壁和隔板可用70％酒精擦拭。
超净台操作野主要用紫外线消毒。
2、器械清洗（1）玻璃器皿的清洗：浸泡→刷洗→浸酸→冲洗（详见课件）（2）塑料器皿冲洗反复利用塑料器皿需经清水浸泡后流水冲洗；流水冲洗→晾干→2％氢氧化钠浸泡过夜→清水冲洗→2～5％盐酸浸泡30min→清水冲洗→蒸馏水漂洗3遍→晾干后包装，以备灭菌。
（3）胶塞等橡胶类用品的处理新胶塞（含有大量滑石粉）：用清水冲洗→ 2％NaOH煮沸15min→清水冲洗→2～5％盐酸煮沸15min→清水冲洗5次以上→蒸馏水漂洗5遍以上→蒸馏水煮沸10min →倒掉废水，余热烘干胶塞备用（或经101.3 kPa高压灭菌）；旧胶塞：用清水浸泡→ 2％NaOH煮沸10～20min →清水冲洗→1％盐酸煮沸30min→清水和蒸馏水漂洗5遍以上→蒸馏水煮沸10min →倒掉废水，余热烘干胶塞备用（也可经101.3 kPa高压灭菌）。
（4）金属器械的清洗新购金属器械：汽油纱布擦去油脂，清水冲洗→酒精棉擦拭，晾干；用过的金属器械：清水煮沸消毒→擦拭干净→包装好高压灭菌消毒。
细胞培养实验
每周甲醛、乳酸、过氧乙酸熏蒸（2小时）和每月 新洁尔灭擦拭地面和墙壁一次的方式进行消毒
生物安全柜
使用前开启柜内紫外灯照射30分钟， 预工作10－15分钟，以除去臭氧和使用工
作台面空间呈净化状态。 使用完毕后，要用70%酒精将台面和台内
四周擦拭干净。 还要定期用福尔马林熏蒸。
细胞培养板
6孔 12孔 24孔 48孔 96孔
细胞培养皿
35mm 60mm 100mm
冻存管
1.2ml 2ml 5ml
离心管
15ml 50ml
细胞刮
滤器
液氮罐
细胞的营养
碳水化合物、氨基酸和脂类三大类
也需要一定量的无机盐、维生素和微量 元素
（四）实验后的要求
关闭超净台风机和电源； 用过的玻璃器材投入清水中浸泡，包装纸叠卷，绳
成束分类归放； 最后用酒精纱布或棉球擦拭工作台面。
细胞培养的基本条件
无菌条件 细胞生长条件 细胞检测条件 细胞保存条件 实验室安全
细胞培养的无菌环境
无菌室的结构：一般由更衣间、缓冲间、操作间三 部分组成。
通常使用浓度为10～15mM
消化液
(1) 胰蛋白酶溶液 主要作用是使细胞间的蛋白质水解，从而使贴壁细胞从瓶壁上脱落并 使细胞游离分散开来 常用浓度为0.25%或5%胰蛋白酶。
(2) EDTA溶液 作用机制是破坏细胞间的连接。 对于一些贴壁特别牢固的细胞，可用EDTA和胰酶的混合液 EDTA溶液的使用浓度为0.02%，配制时应加碱助溶
有限细胞系（finite cell line）：细胞系形成 后仅能维持有限传代次数，最后死亡。