计算溶液所需的质量、体积或浓度。
为什么选择此级别
，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
-80℃储存。特低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
产品介绍：
Ad-mCherry-GFP-LC3B，即adenovirus expressing mCherry-GFP-LC3B fusion protein，是一种可以表达mCherry-GFP-LC3B融合蛋白的腺病毒，可以用于感染细胞或组织后进行细胞自噬(autophagy)的检测。LC3是酵母自噬关键蛋白ATG8在哺乳动物中的同源蛋白。LC3最初被分析鉴定为microtubule-associated protein 1 light chain 3 (MAP1LC3)。LC3蛋白家族包括LC3A、B、C和GABARAP等亚家族，其中对于LC3B的研究最为广泛。到目前为止，LC3B被认为是细胞自噬信号通路最为关键的标志性蛋白。LC3B是一个具有125个氨基酸残基的蛋白，在蛋白合成后，被Atg4所酶切而失去了C端的22个氨基酸蛋白，从而暴露出C端的甘氨酸，人们把它命名为胞质形式的LC3B I。在细胞自噬的过程中，其C端暴露的甘氨酸经过一个类似泛素化的过程，以ATG7为E1样激活酶，以ATG3为E2样连接酶，以Atg12-Atg5-Atg16复合物为E3样连接酶，在C端甘氨酸上共价连接上磷脂酰乙醇胺(phosphatidylethanolamine, PE)而成为LC3B-PE即LC3B II。与胞质定位的LC3B I不同，LC3B II定位于自噬体(autophagosome)的内膜和外膜上。在自噬体与溶酶体融合后，自噬体外膜上的LC3B II被Atg4所酶切，而自噬体内膜上的LC3B II则被溶酶体内的蛋白酶所降解。尽管LC3B II比LC3B I的分子量要大，但由于其极强的疏水性，在SDS-PAGE电泳时，LC3B II比LC3B I迁移得更快，其表观分子量分别为14kD和16kD。自噬(Autophagy)是一种在进化上高度保守的通过溶酶体吞噬并降解部分自身组分的细胞内分解代谢途径。自噬与多种生理功能有关，在饥饿等不利的环境条件下，细胞通过自噬降解多余或异常的细胞内组分，为细胞的生存提供能量及原材料，促进生物体的生长发育、细胞分化及对环境变化产生应答。自噬异常与多种病理过程如肿瘤、神经退行性疾病、代谢疾病、病原体感染等都有密切关系。由于细胞自噬在生理和病理过程中都有重要作用，自噬已经成为细胞生物学领域的一个新的研究热点。Ad-mCherry-GFP-LC3B是阿拉丁自行研发的重组腺病毒，感染后能够在靶细胞中有效表达红色荧光蛋白mCherry、绿色荧光蛋白(GFP)和LC3B的融合蛋白，呈现明亮的红色和绿色荧光，可以用于细胞自噬的检测。自噬小体在与溶酶体融合过程中，溶酶体内的酸性环境会导致GFP荧光淬灭，这为追踪GFP-LC3B的细胞定位增加了难度。mCherry是一种来自于蘑菇珊瑚(mushroom coral)的单体红色荧光蛋白，当其与GFP进行LC3B的共同标记时，在GFP被溶酶体酸性环境所淬灭的情况下，可以发出红色荧光的mCherry因为其卓越的稳定性而被保留。因此，通过融合表达mCherry-GFP-LC3B蛋白，可以非常有效地追踪自噬过程。在用Ad-mCherry-GFP-LC3B腺病毒感染细胞后，在非自噬的情况下，荧光显微镜下mCherry-GFP-LC3B以弥散的黄色荧光(mCherry和GFP的综合效果)形式存在于细胞质中；而在自噬的情况下，荧光显微镜下mCherry-GFP-LC3B则聚集在自噬体膜上，以黄色斑点的形式表现出来(LC3B dot or punctae)；当自噬体与溶酶体融合后，因GFP荧光的部分淬灭而以红色斑点的形式表现出来。腺病毒感染细胞后为瞬时表达，通常不会与基因组DNA重组，不能用于筛选稳定细胞株。本产品感染体内外细胞后，有效表达时间通常不少于7天，不同细胞和组织的有效表达时间有所不同。感染10-14天后融合蛋白的表达很可能会大大减弱。阿拉丁的Ad-mCherry-GFP-LC3B采用了成熟的E1缺陷型重组腺病毒载体系统，感染普通的细胞后不能进行扩增和重组，从而可以有效降低本产品在活体生物中的风险。本产品可以在表达E1的HEK293A、HEK293等适当细胞中扩增。本产品的滴度≥1×108pfu/ml，使用时可以按照108pfu/ml进行计算。如果按照20 MOI感染6孔板的细胞，每孔50万细胞计算，每毫升本产品共可以感染10个孔；如果按照20 MOI感染24孔板的细胞，每孔10万细胞计算，每毫升本产品共可以感染50个孔。如果MOI值提高，则相应可以感染的孔数会减少；如果MOI值降低，则相应可以感染的孔数会增加。
注意事项：
反复冻融会降低病毒滴度，如有必要请在收到本产品后分装保存。病毒融解后，如果在一周内使用，可以放置于4℃。如果-80℃保存时间超过一年，可能会导致滴度下降，此时建议重新测定病毒滴度。腺病毒感染需要细胞表面的CAR受体，请确认拟使用的细胞或组织可以被腺病毒感染。腺病毒对不同细胞的感染效率请参考下表。 对于难感染的细胞，建议使用较高的MOI值(可以达到100、200或500MOI)同时使用促病毒感染试剂Polybrene (终浓度2~4μg/ml)以提高腺病毒的感染效率。本产品使用前请仔细阅读附录1《腺病毒使用安全规范》。本产品生物安全等级为Biosafety Level 1 (BSL-1)，没有确凿证据显示会导致健康成人产生疾病(Not known to consistently cause diseases in healthy adults)，可以按照常规的微生物实验操作要求进行操作。本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。为了您的安全和健康，请穿实验服并戴一次性手套操作。
使用说明：
1.感染条件的确定：不同种类的细胞感染所需的MOI值是不同的，如果是初次使用腺病毒感染需要通过实验确定最佳的感染条件。a.细胞培养(以6孔板NIH3T3细胞为例，其它培养板或培养皿参考6孔板进行操作)：感染前一天在6孔板中以5×105/孔接种NIH3T3细胞，每孔加入2ml完全培养液(具体的接种数量由细胞大小和细胞生长速度而定)，使第二天病毒感染时细胞密度达到约50%左右。b.按照MOI 分别为2、5、10、20、40，计算所需病毒量，计算方法请参考附录2。c.冰上解冻病毒，混匀后备用。d.从细胞培养箱中取出6孔板，在显微镜下确定每孔的细胞均生长良好。吸弃旧培养液，每孔加入1.2ml新鲜培养液，并分别加入特定MOI值的病毒液，同时设置未加入病毒的细胞孔作为对照组。须特别注意在病毒感染时加入尽量少的新鲜培养液，以提升病毒的感染效率。对于培养液体积比较小的多孔板，例如96孔板和48孔板，也可以先把病毒用培养液稀释至所需的MOI值后再加入。e.感染后约24h，除去含有病毒的培养液，每孔加入2ml新鲜的完全培养液，继续培养24h后观察细胞生长状况及荧光蛋白表达情况(参考图1)，以不显著影响细胞生长、荧光较强且感染效率便于荧光观察的MOI值和感染后时间为最佳条件。注1：检测细胞自噬时并非感染效率越高越好，通常约20-70%的感染效率已经足以进行自噬检测，感染效率过高反而容易产生细胞毒性而干扰检测。注2：通常在腺病毒感染细胞后24小时即可观察到荧光蛋白表达的出现，在48小时左右有较强的表达效果。注3：对于感染腺病毒后出现较强细胞毒性的情况，可以尝试在感染后6-12h更换成新鲜的完全培养液。 图1.Ad-mCherry-GFP-LC3B感染细胞的效果图。A、B、C、D、E和F依次为Ad-mCherry-GFP-LC3B分别在MOI值为0、2、5、10、20和40时感染NIH3T3细胞48h后的实拍效果图。上侧为明场照片，下侧为相同视野的荧光照片。2.感染细胞、诱导自噬和荧光观察 按照步骤1. 获得的条件进行感染实验，适当条件诱导自噬(如EBSS或者细胞自噬激活剂处理等)，随后可在荧光显微镜下观察LC3B的荧光变化情况。附录：1.腺病毒使用安全规范 a.作为一种相对安全的病毒，尽管腺病毒基因组在感染后不会整合进入宿主细胞基因组中，也不会在细胞体内进行复制，但是仍然具有可能的潜在生物学危险。我们建议使用者在病毒操作前应仔细阅读本规范，并在实验中严格按照本规范的要求进行操作。更为严格的美国CDC的生物安全等级及其操作与防护要求参考附表1，也可以访问如下网页：https://www.cdc.gov/labs/pdf/CDC-BiosafetyMicrobiologicalBiomedicalLaboratories-2020-P.pdf。 b.腺病毒操作时应使用相应级别的生物安全柜，不同的腺病毒的生物安全等级会有所不同。如果使用普通超净工作台操作病毒，请不要打开排风机，以尽量避免可能污染病毒的尘埃正面吹向操作人员而被吸入。 c.实验操作时必需佩戴一次性帽子、口罩，穿戴实验手套及专门的实验服，避免身体直接接触病毒。手部及面部有开放性创口时，禁止进行病毒操作。 d.操作病毒时需小心谨慎，不要产生气雾或飞溅。如果操作时超净工作台或其它器皿上有病毒污染，请立即用70%乙醇或2%SDS溶液擦拭干净，或者采取其它的妥善措施。 e.如果需要离心，应使用密封性好的离心管，或用封口膜密封后离心，最好使用专门的离心机。 f.用显微镜观察细胞感染情况时应遵从以下步骤：拧紧培养瓶或盖紧培养板，用70%乙醇清理培养瓶或培养板外壁后到显微镜处观察拍照。离开显微镜实验台之前，用70%乙醇擦洗显微镜实验台。 g.所有被病毒污染过的枪头、离心管、培养板(皿、瓶)、培养液、手套等，在丢弃前请用84消毒液或2%SDS浸泡过夜。 h.脱掉手套后，用肥皂或洗手液清洗双手。2.病毒MOI的计算 MOI (Multiplicity of Infection)定义：病毒感染细胞时，病毒数量与细胞数量的比值。 Pfu (Plaque forming units)定义：具有生物活性的病毒颗粒数量。 可以根据如下公式计算所需pfu： 所需pfu=细胞数量×MOI 例如，需要向1×105细胞中加入2 MOI的病毒，即所需pfu=(1×105 cells)×(2 MOI)=2×105 pfu。若病毒母液滴度为1×108 pfu/ml，则细胞培养液中应加入(2×105 pfu)/(1×108 pfu/ml)=0.002ml病毒母液，即2μl病毒母液。 附表1. 生物安全等级及其操作与防护要求Table 1. Summary of Recommended Biosafety Levels for Infectious Agents BSL Agents Practices Primary Barriers andSafety Equipment Facilities(Secondary Barriers) 1 Not known to consistently cause diseases in healthy adults Standard microbiological practices ■ No primary barriers required.■ PPE: laboratory coats and gloves; eye, face protection, as needed Laboratory bench and sink required 2 ■ Agents associated with human disease■ Routes of transmission include percutaneous injury, ingestion, mucous membrane exposure BSL-1 practice plus: ■ Limited access■ Biohazard warning signs■ “Sharps” precautions■ Biosafety manual defining any needed waste decontamination or medical surveillance policies Primary barriers:■ BSCs or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials■ PPE: Laboratory coats, gloves, face and eye protection, as needed BSL-1 plus:■ Autoclave available 3 Indigenous or exotic agents that may cause serious or potentially lethal disease through the inhalation route of exposure BSL-2 practice plus: ■ Controlled access■ Decontamination of all waste■ Decontamination of laboratory clothing before laundering Primary barriers:■ BSCs or other physical containment devices used for all open manipulations of agents■ PPE: Protective laboratory clothing, gloves, face, eye and respiratory protection, as needed BSL-2 plus:■ Physical separation from access corridors■ Self-closing, double-door access■ Exhausted air not recirculated■ Negative airflow into laboratory■ Entry through airlock or anteroom■ Hand washing sink near laboratory exit 4 ■ Dangerous/exotic agents which post high individual risk of aerosol-transmitted laboratory infections that are frequently fatal, for which there are no vaccines or treatments■ Agents with a close or identical antigenic relationship to an agent requiring BSL-4 until data are available to redesignate the level■ Related agents with unknown risk of transmission BSL-3 practices plus:■ Clothing change before entering■ Shower on exit■ All material decontaminated on exit from facility Primary barriers:■ All procedures conducted in Class III BSCs or Class I or II BSCs in combination with full-body, air-supplied, positive pressure suit BSL-3 plus:■ Separate building or isolated zone■ Dedicated supply and exhaust, vacuum, and decontamination systems■ Other requirements outlined in the text BSL, biosafety level; PPE, personal protective equipment.
Aladdin's Ad-mCherry-GFP-LC3B is an adenovirus that can express the mCherry-GFP-LC3B fusion protein in infected cells or tissues, which can be used for autophagy assays following infection of cells or tissues.Autophagy is a highly conserved intracellular process that degrades long-lived proteins, organelles, and other cytoplasmic components via the lysosomal pathway. Under unfavorable conditions such as starvation, cells degrade excess or abnormal intracellular components through autophagy to provide energy and raw materials for cell survival, promoting growth and development of organisms, cell differentiation, and responses to environmental changes. Abnormal autophagy is closely related to a variety of pathological processes such as tumors, neurodegenerative diseases, metabolic diseases, and pathogen infections. Because of the importance of autophagy in both physiological and pathological processes, it has become a new hot research topic in the field of cell biology.LC3 is the mammalian homolog of ATG8 which is a key protein in yeast autophagy. The LC3 protein family includes LC3A, LC3B, LC3C and GABARAP subfamilies, among which LC3B is the most extensively studied. LC3B has been considered to be the most critical marker protein in the autophagy signaling pathway. The pro-form of LC3B consisting of 125 amino acids is cleaved by Atg4 into the LC3B-I form with 22 amino acids lost at the C-terminus, thus exposing a C-terminal glycine. In the process of autophagy, LC3B-I undergoes a ubiquitination-like process and becomes the LC3B-II form with phosphatidylethanolamine (PE) covalently linked with the C-terminal glycine, during which ATG7 acts as the E1-like activating enzyme, ATG3 as the E2-like conjugation enzyme, and the Atg12-Atg5-Atg16 complex as the E3-like ligase. Unlike LC3B-I which is localized in cytoplasm, LC3B-II is localized on the inner and the outer membranes of autophagosome. After autophagosome-lysosome fusion, LC3B-II on the outer membrane of autophagosome is cleaved by Atg4, while LC3B-II on the inner membrane is degraded by proteases in lysosome. Although LC3B-II has a larger molecular weight than LC3B-I, it migrates faster than LC3B-I during SDS-PAGE electrophoresis due to its extreme hydrophobicity, appearing 14kD and 16kD, respectively.Ad-mCherry-GFP-LC3B is a recombinant adenovirus developed by aladdin, which can effectively express LC3B proteins fused with red fluorescent mCherry as well as green fluorescent GFP in infected cells.In the process of autophagosome-lysosome fusion, the fluorescence of GFP will be quenched due to the acidic environment within lysosome, making it difficult to track the intracellular location of GFP-LC3B. However, mCherry is a monomeric fluorescent protein from mushroom coral and its red fluorescence is stable under acidic conditions. Therefore, the mCherry-EGFP-LC3B fusion protein enables effective tracking of autophagy processes. After infecting cells with the Ad-mCherry-GFP-LC3B adenovirus, non-autophagy cells exhibit diffusive yellow fluorescence in cytoplasm due to the combination of green and yellow fluorescence, while in cells with autophagy, mCherry-GFP-LC3B proteins aggregate on the autophagosome membrane and appear as yellow spots (LC3B dot or punctae) or as red spots due to partial quenching of GFP fluorescence during the process of autophagosome-lysosome fusion.The adenovirus does not integrate the target gene into genomic DNA of infected cells, thus mediating transient protein expression only. The effective expression time after infection of cells in vitro and in vivo is usually no less than 7 days, which may vary depending on the types of cells and tissues. The expression level of the fusion protein is likely to be greatly reduced after 10-14 days of infection.Aladdin's Ad-mCherry-GFP-LC3B is a mature E1-deficient recombinant adenovirus which cannot replicate or recombine in ordinary cells, thus effectively reducing the risk of this product in living organisms.This product can be amplified in appropriate cells such as HEK293A and HEK293 expressing E1.The titer of this product is over 1×108 pfu/ml and can be used as 108 pfu/ml. MOI (Multiplicity of Infection) is the ratio of the number of virus to the number of cells when virus infects cells. When 500,000 cells per well of 6-well plates are infected at 20MOI, each ml of this product can infect a total of 10 wells; when 100,000 cells per well of 24-well plates are infected at 20 MOI, each ml of this product can infect 50 wells in total. The MOI value can be adjusted and the number of wells that can be infected by this product will change correspondingly.
Precautions：
Repeated freeze-thaws will reduce the viral titer. If necessary, please store in aliquots at the first use. Aliquoting must be performed on ice. This product can be stored at 4℃ if it is used within a week, but it should be noted that the viral titer decreases over the time of storage at 4℃. If stored at -80℃ for more than one year, the titer may decrease, and it is recommended to re-titrate this product before use.Adenovirus infection requires the presence of CAR receptors in cell surface. Please ensure that the cells or tissues to be infected possess CAR receptors on the cell surface.Please refer to the table below for the infection efficiency of adenovirus on different cells.SpeciesCell lineOriginTransduction efficiencyHuman293Embryonic Kidney95%MCF-10AMammary epithelium51%MDA-MB-231Malignant breast cancer cell72%HepG2Hepatocarcinoma70%T24Transitional cell carcinoma37%TCCSUPTransitional cell carcinoma53%PC-3Prostate cancer metastases site67%DU145Prostate cancer metastases site86%LNCaPProstate cancer metastases site69%JurkatT cell leukemia2%HRTRectal tumor5%MurineMT1A2Mammary tumor transformed with SV40 T antigen92%NIH 3T3Mouse fibroblast12%WEHIFibrosarcoma10%AML12Liver28%MS-K68Spleen48%EL4T cell2%BovineFBRT HE1Fetal retina transformed with Had5 E190%MDBKKidney70%FBK-34Kidney transformed with BAd3 E125%BT-5705Turbinate28%EBLEmbryonic lung8%PorcineFPRT HE1-5Fetal retina transformed with HAd5 E191%PK-15Kidney76%PK-M12Kidney cell transformed with SV40 T antigen71%PT-K75Turbinate22%EPLEmbryonic lung53%SBC-1765Derived from buffy coat cells73%For cells that are difficult to transduct, it is recommended to use a higher MOI (up to 100, 200 or 500 MOI) and Polybrene at a final concentration of 2~4 μg/ml to improve the infection efficiency of adenovirus.Please read Appendix 1 "Safety Specifications for Lentivirus Use" carefully before using this product. The biosafety level of this product is Biosafety Level 1 (BSL-1. It is not known to consistently cause diseases in healthy adults and can be handled with standard microbiological practices.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use：
1.Determination of infection conditionsMOI (Multiplicity of Infection) definition: the ratio of the number of viruses to the number of cells when the virus infects cells. The MOI values required for different types of cells are different. For the first use of adenovirus, it is necessary to optimize the infection conditions through experiments. The following protocol is provided for NIH3T3 cells cultured in 6-well plates. For cells cultured in other types of vessels, the following protocol can be adjusted appropriately.a.One day before infection, inoculate 5×105 cells in 2ml of complete culture medium per well of a 6-well plate. Cells should be about 50% confluent at the time of virus infection. Note: The specific cell number of inoculations depends on cell type, cell size, cell growth rate and other factors.b.Referring to Appendix 2, calculate the required amount of virus to yield the MOI values of 2, 5, 10, 20 and 40, respectively.c.Thaw the virus on ice and mix well before use.d.Take the 6-well plate from the incubator and observe the cells under microscopy to make sure the cells are in good growth state. Replace the old culture medium with 1.2ml of fresh culture medium, and pipette appropriate amount of virus, calculated in step b, into appropriate wells. Meanwhile, set up the wells without virus as negative control. Special attention should be paid to adding as little fresh culture medium as possible to increase the infection efficiency of virus. For multi-well plates with small volume of culture medium, such as 96-well plates and 48-well plates, the virus can be diluted with culture medium to the desired MOI value before addition.e.About 24 hpi (hours post-infection), replace the virus-containing medium with 2ml of fresh complete medium to each well, and continue to culture cells for 24h, then check the cell growth state and the expression level of fluorescent proteins under a fluorescence microscopy (Figure 1). The optimal MOI value and post-infection time are those under which cell growth is relatively good, with high GFP expression and high infection efficiency of virus. Note 1: An infection efficiency of 20-70% is usually sufficient for autophagy assays. An excessively high infection efficiency is likely to cause cytotoxicity and interference with the assay.Note 2: Usually the fluorescent protein expression can be visible after 24 hours of adenovirus infection, with higher expression at about 48 hpi.Note 3: In case of strong cytotoxicity after adenovirus infection, replacing the virus-containing medium with fresh complete culture medium after 6-12 hpi can be attempted.Figure 1. Expression of mCherry-GFP-LC3B fusion proteins in NIH3T3 cells infected with ’s Ad-mCherry-GFP-LC3B . A, B, C, D, E and F are images of NIH3T3 cells infected with Ad-mCherry-GFP-LC3B at MOI values of 0, 2, 5, 10, 20 and 40 for 48h, respectively. Images taken under bright field (upper panel) and images of the same view under GFP channel (middle panel) and mCherry channel (lower panel).2.Observation of infected cells, induced autophagy and fluorescence After performing virus infection experiments using conditions determined in step 1, induce autophagy with EBSS or other appropriate inducers, and then observe the changes of fluorescence under a fluorescence microscope.Appendix:1.Safety specifications for handling adenovirus:a.As a relatively safe virus, although the adenovirus genome does not integrate into the host cell genome or replicate in cells after infection, it still has potential biological risks. We recommend that users read the specifications carefully before virus manipulation and operate in strict accordance with the requirements. For more stringent US CDC biosafety levels and their operation and protection requirements, please refer to Appendix 1 or visit the following website: http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_sect_IV.pdf.b.Biosafety cabinets of corresponding levels should be used for adenovirus operations, and the biosafety levels vary for different adenoviruses. When using an ordinary laminar flow hood to operate the virus, please do not turn on the exhaust fan to avoid the dust that may be virus-contaminated being blown to the operator and inhaled.c.Disposable hats, masks, laboratory gloves and special laboratory coats must be worn during the experiments to avoid direct contact with the virus. Virus manipulation is prohibited when there are open wounds on the hands and face.d.Be careful not to produce aerosol or splash when handling the virus. If the the laminar flow hood or other utensils are contaminated by the virus during operation, please wipe them with 70% ethanol or 2% SDS solution immediately, or take other appropriate measures.e.If centrifugation is required, use a well-sealed centrifuge tube, or seal it with parafilm and centrifuge, preferably using a centrifuge designated for virus operation.f.The following steps should be followed when observing cell infection under a microscope: Tighten the culture flask or cover the culture plate, clean the outer surface of the culture flask or culture plate with 70% ethanol, and then examine by microscope. After finishing examination, clean the microscope bench with 70% ethanol.g.All virus-contaminated pipette tips, centrifuge tubes, culture plates (dishes, bottles), culture solution, gloves, and other consumables should be soaked in disinfectant or 2% SDS overnight before discarding.h.After removing gloves, wash hands with soap or hand sanitizer.2.Calculation of virus MOI:MOI (Multiplicity of Infection) is defined as the ratio of the number of viruses to the number of cells when virus infects cells.Pfu (plaque forming units) defines the number of biologically active virus particles.The required pfu can be calculated according to the following formula.Required pfu = number of cells x MOIFor example: If 2 MOI of virus is needed for infecting 1 × 105 cells, the required pfu = (1 × 105 cells) × (2 MOI) = 2 × 105 pfu. For a virus stock with a titer of 1×108 pfu/ml, the volume of the virus stock required will be 2×105 pfu / (1×108 pfu/ml) × 1000 = 2μl.Table 1. Biosafety levels and their operation and protection requirements.BSLAgentsPracticesPrimary Barriers andSafety EquipmentFacilities(Secondary Barriers)1Not known to consistently cause diseases in healthy adultsStandard microbiological practices■ No primary barriers required.■ PPE: laboratory coats and gloves; eye, face protection, as neededLaboratory bench and sink required2■ Agents associated with human disease■ Routes of transmission include percutaneous injury, ingestion, mucous membrane exposureBSL-1 practice plus: ■ Limited access■ Biohazard warning signs■ “Sharps” precautions■ Biosafety manual defining any needed waste decontamination or medical surveillance policiesPrimary barriers:■ BSCs or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials■ PPE: Laboratory coats, gloves, face and eye protection, as neededBSL-1 plus:■ Autoclave available3Indigenous or exotic agents that may cause serious or potentially lethal disease through the inhalation route of exposureBSL-2 practice plus: ■ Controlled access■ Decontamination of all waste■ Decontamination of laboratory clothing before launderingPrimary barriers:■ BSCs or other physical containment devices used for all open manipulations of agents■ PPE: Protective laboratory clothing, gloves, face, eye and respiratory protection, as neededBSL-2 plus:■ Physical separation from access corridors■ Self-closing, double-door access■ Exhausted air not recirculated■ Negative airflow into laboratory■ Entry through airlock or anteroom■ Hand washing sink near laboratory exit4■ Dangerous/exotic agents which post high individual risk of aerosol-transmitted laboratory infections that are frequently fatal, for which there are no vaccines or treatments■ Agents with a close or identical antigenic relationship to an agent requiring BSL-4 until data are available to redesignate the level■ Related agents with unknown risk of transmissionBSL-3 practices plus:■ Clothing change before entering■ Shower on exit■ All material decontaminated on exit from facilityPrimary barriers:■ All procedures conducted in Class III BSCs or Class I or II BSCs in combination with full-body, air-supplied, positive pressure suitBSL-3 plus:■ Separate building or isolated zone■ Dedicated supply and exhaust, vacuum, and decontamination systems■ Other requirements outlined in the textBSL, biosafety level; PPE, personal protective equipment.
规格
产品名称
Ad-mCherry-GFP-LC3B
别名
腺病毒载体
分子类型
蛋白质
产品线
生物产品
储存与运输
储存条件
-80℃储存
运输条件
特低温运输
稳定性与储存
-80℃保存，一年有效。-20℃保存，1-2个月内有效。4℃保存，一周内有效。
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