| Product Description | 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.Related Products:Cat. 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