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
Lenti-mCherry-EGFP-LC3B，即Lentivirus expressing mCherry-EGFP-LC3B fusion protein，是一种可以表达mCherry-EGFP-LC3B融合蛋白的慢病毒，可以用于感染细胞或组织后进行细胞自噬(autophagy)检测。本产品带有嘌呤霉素(puromycin)抗性，感染细胞后可以筛选稳定株以用于后续的自噬研究。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。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。Lenti-mCherry-EGFP-LC3B是阿拉丁自行研发的重组慢病毒，感染后能够在靶细胞中有效表达红色荧光蛋白(mCherry)、增强型绿色荧光蛋白(EGFP)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。本产品感染HEK293T细胞的效果如图1所示。图1. Lenti-mCherry-EGFP-LC3B感染HEK293T细胞的效果图。96孔板每孔10,000个细胞，培养过夜后用相应的病毒TU数进行感染，病毒感染72h后荧光显微镜实拍相同视野效果图。实际检测效果会因检测仪器、实验条件的不同而存在差异，本图仅供参考。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EGFP荧光淬灭，这为追踪EGFP-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EGFP进行LC3B的共同标记时，在EGFP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-EGFP-LC3B蛋白，可以非常有效地追踪自噬过程。在用Lenti-mCherry-EGFP-LC3B慢病毒感染细胞后，在非自噬的情况下，荧光显微镜下mCherry-EGFP-LC3B以弥散的黄色荧光(mCherry和EGFP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-EGFP-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EGFP荧光的部分淬灭而以红色斑点的形式表现出来。慢病毒感染细胞后会将目的基因随机整合到基因组DNA上，因而可以长期稳定地表达目的蛋白，并且几乎可以感染所有哺乳动物细胞，在很多不分裂的细胞中也可以长期稳定表达，广泛应用于细胞和动物实验。本产品表达嘌呤霉素抗性基因，因此本产品感染培养的细胞后可以使用嘌呤霉素筛选稳定表达的细胞株以用于后续研究。阿拉丁的Lenti-mCherry-EGFP-LC3B是复制缺陷型。其3’ LTR的增强子功能发生缺失，形成了自失活(self-inactivating) 3’ LTR，并且5’ LRT中的U3区域替换成CMV启动子，在感染普通的细胞后不能进行复制和扩增，从而有效降低了本产品在活体生物中的风险。阿拉丁推荐的慢病毒感染不同种类体外培养细胞的MOI值参见下表。实验室常用的逆转录病毒(Retrovirus)、慢病毒(Lentivirus)、腺相关病毒和腺病毒(Adenovirus)的主要特征之间的比较和差别参见下表。具体的特定病毒的一些特征和下表相比可能会有一定差异。阿拉丁提供慢病毒、腺病毒和腺相关病毒三种类型用于自噬检测的产品：Lenti-mCherry-EGFP-LC3B 、Lenti-EGFP-LC3B 、Ad-mCherry-GFP-LC3B 和AdPlus-mCherry-GFP-LC3B 、AAV-mCherry-GFP-LC3B 。Lenti-mCherry-EGFP-LC3B 和Lenti-EGFP-LC3B 适合用于一些腺病毒较难感染或者需要筛选稳定细胞株的情况；Ad-mCherry-GFP-LC3B 和AdPlus-mCherry-GFP-LC3B 由于表达起始时间短，适合用于能被腺病毒感染并且希望快速获得实验结果的情况，也可以在表达E1的HEK293A、HEK293等适当细胞中扩增，便于后续长期使用；AAV-mCherry-GFP-LC3B 由于其组织特异性和低免疫原性更适合用于动物实验。四种产品的比较和选择请参见下表。本产品的滴度为10^9TU/ml，适合细胞实验或活体动物实验。TU, transduction unit，即转导单位。TU通过本产品梯度稀释后感染HEK293T细胞后测定获得，能让一个HEK293细胞呈现绿色荧光的活力单位定义为1TU。MOI (Multiplicity of Infection)是病毒感染细胞时，病毒数量与细胞数量的比值。使用10^9TU/ml的本产品，如果按照5 MOI感染6孔板的细胞，每孔50万细胞计算，100μl共可以感染40个孔；如果按照5 MOI感染24孔板的细胞，每孔10万细胞计算，100μl共可以感染200个孔。如果MOI值提高，那么相应可以感染的孔数会减少；如果MOI值下调，那么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。分装时必须在冰浴上进行。病毒融解后，如果在一周内使用，可以放置于4℃，但须注意4℃存放时间越长，滴度下降越明显。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。本产品使用前请仔细阅读附录1《慢病毒使用安全规范》。本产品生物安全等级为Biosafety Level 2 (BSL-2)，在按照常规的微生物实验操作要求进行操作的基础上，还需要注意限制接触、生物危害提示、显著的警示标识、并制定相应的安全规范。病毒操作中应注意有效防护，绝对禁止在生物安全柜内有任何皮肤直接暴露的情况。实验完成后，请及时清洗双手。严禁直接接触病毒，如意外接触，请及时用清水冲洗，并适当用70%乙醇对皮肤进行消毒。任何接触过病毒的材料、试剂、样品，应经消毒处理，可以采用1%的SDS溶液、或84消毒液(1:20)浸泡30分钟以上，或121℃高压灭菌30分钟。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：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。TU (Transduction Units)定义：具有生物活性的病毒颗粒数量。梯度稀释病毒后感染细胞，根据出现荧光的细胞数量来确定具有生物活性的病毒颗粒数量。不同种类的细胞感染所需的MOI值是不同的，如果是初次使用慢病毒感染需要通过实验确定最佳的感染条件。理论上MOI值越高，慢病毒感染可能性越大，感染效率越高，对细胞的毒性也越大。所以预实验的目的是在保证细胞成活率的情况下，确定能够使感染效率达到适宜观察水平的MOI值。a.细胞培养(以24孔板HEK293T细胞为例，其它培养板或培养皿参考24孔板进行操作)：感染前一天，在24孔板中以约5×104/孔接种HEK293T细胞，每孔加入0.5ml完全培养液，使第二天病毒感染时的细胞密度达到约70-80% (细胞数约1×105)。注：具体接种数量需根据细胞种类、细胞大小和细胞生长速度等因素而确定。b.设置MOI值分别为1、2、5、10、20，计算所需病毒量，计算公式如下：TU = 感染时的细胞数* × MOI病毒母液(μl) = TU / 滴度(TU/ml) × 1000*一般第二天细胞数按接种细胞数增长1倍来计算，增殖较慢或不增殖的细胞可适当调整。例如：需要向1×105个HEK293T细胞加入20 MOI的病毒，即所需TU = (1×105 cells) × (20 MOI) =2×106 TU。若病毒母液滴度为1×109 TU/ml，则细胞培养液中应加入病毒母液 = 2×106 TU / (1×109 TU/ml) × 1000 = 2μl，即2μl病毒母液。注：其它细胞株的预实验MOI设置可以参考本说明书中产品简介部分“阿拉丁推荐的慢病毒感染不同种类体外培养细胞的MOI值”表格或相关文献资料。c．按照下图设置MOI预实验组，建议设置复孔以确保实验准确性。注：对于适合使用聚凝胺(polybrene)处理的细胞，弃旧培养液，每孔加入250μl含有6-8μg/ml polybrene的新鲜培养液；对于不适合使用polybrene处理的细胞，弃旧培养液，每孔加入250μl的新鲜培养液。使用polybrene可以使感染效率提高约2-10倍，并且病毒测定其活力的时候是使用polybrene的。须特别注意此时宜加入尽量少的新鲜培养液，以提升后续病毒的感染效率。 图2. 细胞感染MOI值预实验设计分组。MOI依次设置为：1、2、5、10、20。并同时设置加含6-8μg/ml polybrene的培养液实验组，不含polybrene的培养液实验组及Blank组(空白对照组)。Blank组可作为参照，以检验细胞生长状态。本图中实验设置仅供参考，用户应根据实际情况自行适当调整。d．取出本产品置于冰上解冻，混匀后，将根据特定MOI值计算好的病毒母液量分别加入细胞培养板中，轻轻混匀后继续培养。注：如果病毒滴度太高，需加的病毒母液量较少或多孔板如96孔板和48孔板等培养液体积比较小的情况，可以先将病毒母液用培养液进行适当稀释后再加入。e．感染后约4小时，每孔补加250μl 新鲜培养液。f．感染后约24小时，进行换液。弃含病毒的培养液，每孔加入新鲜的完全培养液，继续培养。注：换液的具体时间需视细胞状态而定，如果慢病毒对细胞有明显毒性作用，影响细胞生长状态，最短可于加病毒4小时后更换新鲜培养液。g．继续培养约48-96小时后观察细胞生长状况及荧光蛋白表达情况，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。h．如果有必要，可以根据特定细胞加入适当浓度的嘌呤霉素(puromycin) ，以筛选被感染的细胞，后续可以通过稀释法等获取单克隆细胞株。嘌呤霉素的浓度可以通过梯度稀释的预实验来确定，以刚好能完全杀死目的细胞的浓度为宜。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：通常在慢病毒感染细胞后72小时即可观察到荧光蛋白表达的出现，在96小时左右有较强的表达效果。注3：对于感染慢病毒后出现较强细胞毒性的情况，可以尝试在感染后4小时更换成新鲜的完全培养液。2.感染细胞、诱导自噬和荧光观察：按照步骤1获得的条件进行病毒感染实验，在观察到mCherry-EGFP-LC3B的绿色荧光后，或者在筛选获得mCherry-EGFP-LC3B的稳定表达细胞株后，使用Earle's Balanced Salt Solution 或者细胞自噬激活剂处理等适当条件诱导自噬，随后在荧光显微镜下观察LC3B的荧光变化情况。 附录： 1.慢病毒使用安全规范：a.作为一种相对安全的病毒，尽管慢病毒在正常细胞内不能进行复制和扩增，但是慢病毒基因组可以整合到被感染细胞的基因组中，因此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.慢病毒操作时应使用相应级别的生物安全柜，不同的慢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i.病毒飞溅或是含有病毒的气溶胶与人体接触，须用大量清水冲洗眼睛、皮肤或粘膜等接触的部位至少15min。j.含病毒的针头或是其它利器刺破皮肤，伤口应立即用10%的碘伏溶液擦洗数分钟，然后用大量清水冲洗。k.装盛慢病毒的实验用品需单独放置，并须加以适当标示。l.对同实验室的人员须进行慢病毒安全培训或安全警示。2.生物安全等级及其操作与防护要求：Summary of Recommended Biosafety Levels for Infectious Agents BSL Agents Practices Primary Barriers and Safety 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 per-cutaneous injury, ingestion, mucous membrane exposure SBSL-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 Lenti-mCherry-EGFP-LC3B is a recombinant lentivirus that can express mCherry-EGFP-LC3B fusion protein in infected cells or tissues. The stable cells infected by this lentivirus can be selected with puromycin and used for autophagy detection. 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-1 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 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.Lenti-mCherry-EGFP-LC3B is a recombinant lentivirus developed by aladdin. It can effectively express LC3B proteins fused with both red fluorescent mCherry and enhanced green fluorescent EGFP in infected cells, and can be used for autophagy detection. Please refer to Figure 1 for the infected HEK293T cells by this product.Figure 1. Expression of mCherry-EGFP-LC3B fusion proteins in HEK293T cells infected with the Lenti-mCherry-EGFP-LC3B . HEK293T cells cultured in 96-well plates were infected with different transduction units (TUs) as indicated in the figure. After 72 hours post-infection, cells were examined by fluorescence microscope. This figure is for reference only, which may vary due to different experimental conditions.In the process of autophagosome-lysosome fusion, the fluorescence of EGFP will be quenched due to the acidic environment within lysosome, making it difficult to track the intracellular location of EGFP-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 Lenti-mCherry-EGFP-LC3B virus, non-autophagy cells exhibits diffusive yellow fluorescence in cytoplasm due to the combination of green and yellow fluorescence, while in cells with autophagy, mCherry-EGFP-LC3B aggregates on the autophagosome membrane and appears as yellow spots (LC3B dot or punctae) or as red spots due to partial quenching of EGFP fluorescence during the process of autophagosome-lysosome fusion.After cell infection, the lentivirus will randomly integrate the target genes into the genomic DNA to enable a stable expression of target proteins in host cells, even in non-dividing host cells. Therefore, this product has been widely used in cell and animal experiments.Aladdin's Lenti-mCherry-EGFP-LC3B is a replication-deficient lentivirus. The enhancer function of its 3' LTR is lost, forming a self-inactivating 3' LTR, and the U3 region in the 5' LRT is replaced with the CMV promoter. This lentivirus product cannot replicate and proliferate after infecting ordinary cells, thereby effectively reducing the risk of this product in living organisms.The multiplicity of infection (MOI) values recommended for infecting different types of in vitro cultured cells are listed in the table below
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.Please read Appendix 1 "Safety Specifications for Lentivirus Use" carefully before using this product. The biosafety level of this product is Biosafety Level 2 (BSL-2). Besides following the standard microbiological practices, it is also necessary to pay attention to limiting exposure, biohazard reminders, prominent warning signs, and develop appropriate safety regulations.Effective protections should be taken when handling virus, and any direct skin exposure is not allowed. Please wash your hands immediately after the experiment. It is strictly forbidden to directly contact the virus. In case of accidental contact, please rinse with water immediately, and properly disinfect the skin with 70% ethanol.Any materials, reagents, and samples that have come into contact with virus should be disinfected by soaking in 1% SDS solution or 84 disinfectant (1:20) for more than 30 minutes, or sterilized by autoclaving at 121℃ for 30 minutes.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. TU (Transduction Units) Definition: The number of biologically active viral particles. After infecting cells with serial dilutions of the virus, the number of biologically active virus particles was determined based on the number of fluorescent cells or the qPCR assay of extracted cellular genomic DNA. The MOI values required for different types of cells are different, and for the first use of lentivirus, it is necessary to optimize the infection conditions. In theory, the higher the MOI value, the higher the infection efficiency, but with greater cytotoxicity. Therefore, the purpose of the pre-experiment is to determine the MOI value that can make the infection efficiency reach a level suitable for observation while ensuring the cell survival rate.The following protocol is provided for HEK293T cells cultured in 24-well plates. For cells cultured in other types of vessels, the following protocol can be adjusted appropriately.a.One day before infection, inoculate 5×104 cells in 0.5ml of complete culture medium per well of a 24-well plate. Cells should be about 70-80% confluent at the time of virus infection the next day. Note: The specific cell number of inoculations depends on cell type, cell size, cell growth rate and other factors.b.Calculate the required amount of virus to yield the MOI values of 1, 2, 5, 10, and 20, respectively. The calculation formula is as follows:TU = number of cells at infection* × MOIVirus stock solution (μl) = TU / titer (TU/ml) × 1000*Generally, the number of cells on the second day is calculated by doubling the number of inoculated cells, and the cells that proliferate slowly or do not proliferate can be adjusted appropriately.For example: If 20 MOI of virus is needed for infecting 1 × 105 HEK293T cells, the required TU is 2 × 106 TU (1 × 105 × 20). For a virus stock with a titer of 1×109 TU/ml, the volume of the virus stock required will be 2×106 TU / (1×109 TU/ml) × 1000 = 2μl.Note: For a preliminary test of MOI settings for other cell lines, please refer to the table “MOI values recommended by for lentivirus infection of different types of in vitro cultured cells” in the product introduction section of this manual or related literature.c.Set up the MOI preliminary test groups according to the figure below. It is recommended to run in duplicate to ensure the accuracy of the experiment. Note: For cells that can be treated with polybrene (, C0351/ST1380), replace the old culture medium with 250μl of fresh culture medium containing 6-8μg/ml polybrene per well; for cells not suitable for treatment with polybrene, discard the old medium and add 250μl of fresh medium to each well. The use of polybrene can increase the infection efficiency by about 2-10 times, and the viability of the virus is determined using polybrene. At this step, fresh culture medium should be added as less as possible to improve the infection efficiency subsequently.Figure 2. Grouping of preliminary tests to determine the optimal MOI value for cell infections. The MOIs were set to be 1, 2, 5, 10, 20, as indicated. Experimental groups with or without 6-8μg/ml polybrene and their corresponding blank groups without virus infection were designed. Blank groups were used to check the cell growth state. The experimental settings in this figure are for reference only, and can be adjusted appropriately based on the experimental requirements.d.Thaw this product on ice, mix well, and pipette appropriate amount of virus, calculated in step b, into appropriate wells. Mix gently and continue to culture. Note: When the needed amount of virus stock solution is too small, dilute the virus stock solution appropriately with culture medium before adding to plates.e.After 4 hours post-infection (hpi), add 250μl of fresh culture medium to each well.f.After 24 hpi, replace the virus-containing medium with fresh complete medium to each well, and continue to culture cells. Note: The time for changing culture medium depends on the growth state of cells. If the lentivirus is toxic to cells obviously and affects their growth, the medium can be replaced after 4 hpi.g.After culturing for another 48-96 hours, check the cell growth state and the expression level of fluorescent protein. The optimal MOI value and post-infection time are those under which cell growth is relatively good, with high fluorescent protein expression and high infection efficiency.h.If necessary, an appropriate concentration of puromycin (, ST551) can be added to select for infected cells, and then the monoclonal cell line can be obtained by dilution method. The concentration of puromycin can be determined by preliminary tests of serial dilutions of puromycin, and the concentration that can just kill the target cells completely is considered to be appropriate.Note 1: The infection efficiency is not necessarily the higher the better for autophagy assay, because too high infection efficiency could cause cytotoxicity. The infection efficiency of 20-70% is generally sufficient.Note 2: Fluorescent protein can be visible usually after 72 hpi, with higher expression at about 96 hpi.Note 3: In cases where strong cytotoxicity occurs after infection with lentivirus, replacing the virus-containing medium with fresh complete culture medium after 4 hpi can be attempted.2.Observation of infected cells, induced autophagy and fluorescencePerform virus infection experiments using the conditions determined in step 1. After the green fluorescence of mCherry-EGFP-LC3B is visible, or after obtaining the cell lines stably expressing the mCherry-EGFP-LC3B, use Earle's Balanced Salt Solution (, C0213/C0214) or appropriate inducers to induce autophagy, and then observe the changes of fluorescence under a fluorescence microscope.Appendix:1.Safety specifications for handling lentivirus:a.As a relatively safe virus, although lentivirus cannot replicate and proliferate in normal cells, the lentivirus genome can integrate into the genome of infected cells, so it still has possible potential biological dangers. We suggest that users should read the specifications carefully before virus operation, and operate in strict accordance with the requirements of the specifications. For more stringent US CDC biosafety levels and their operation and protection requirements, please refer to Appendix 1, or visit the following webpage: https://www.cdc.gov/labs/pdf/CDC-BiosafetyMicrobiologicalBiomedicalLaboratories-2020-P.pdf.b.Biosafety cabinets of corresponding levels should be used for lentivirus operations, and the biosafety levels of different lentiviruses will vary. When using an ordinary laminar 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 laminar hood or other utensils are contaminated by the virus during operation, please clean 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 with 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 finish examining, 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.i.When virus splashes or virus-containing aerosols contact with eyes, skin or mucous membranes, immediately wash with plenty of water for at least 15 minutes.j.If needles or other sharp instruments containing virus pierce the skin, the wound should be immediately scrubbed with 10% iodophor solution for several minutes, and then rinsed with plenty of water.k.The experimental supplies containing lentiviruses should be kept separately and properly marked.l.Lentivirus safety training or safety warnings must be given to personnel in the same laboratory.2.Biosafety levels and their operation and protection requirements:BSLAgentsPracticesPrimary Barriers and Safety 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 per¬cutaneous 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 laundering Primary barriers:■ BSCs or other physical containment devices used for all open manipula¬tions 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-trans¬mitted laboratory infections that are frequently fatal, for which there are no vaccines or treatments■ Agents with a close or identical anti¬genic relationship to an agent requir¬ing 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 com¬bination 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
Lenti-mCherry-EGFP-LC3B
别名
慢病毒载体mCherry-EGFP-LC3B
规格或纯度
10^9TU/ml
分子类型
基因
产品线
生物产品
储存与运输
浓度
10^9TU/ml
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。
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
技术文档和文章
溶液计算器
常见问题
本产品如何运输？
本产品采用干冰配冷袋冷冻运输。收货后请立即拆包，并按上述储存条件保存；处理干冰时请戴隔热手套并保持通风。
本产品应如何储存？
请于?80 °C条件下保存。需超低温保存以维持规定的有效期。
如何获取批次专用COA？
每个订单都会随附分析证书（COA），您也可以从账户订单历史中下载。如需订购前的COA，请联系您的客户代表，或使用PDP上的"申请COA"按钮——我们将提供代表性批次的COA。
产品仅提供给经过验证的企业、机构和合格的专业人员用于研发。不适用于人类、动物、诊断或治疗。
Copyright © 2023至今 上海阿拉丁生化科技股份有限公司 保留所有权利。
危险品化学品经营许可证（带存储）