| Product Description | Ad-mCherry-p62 (adenovirus expressing mCherry-p62 fusion protein) is a recombinant adenovirus developed by aladdin, which can express red fluorescent mCherry-p62 fusion protein high efficiently in target cells or tissues after infection. It is widely used for the detection of autophagy in target cells or tissues exhibiting the bright red fluorescence.Ad-mCherry-p62 does not recombine with genomic DNA of target cells or tissues, and the expression of mCherry-P62 fusion protein is transient. Therefore, it cannot be used to screen stable cell lines. After infection of cells, the expression of mCherry-P62 can last for at least 7 days usually and decreases significantly after 10-14 days, which may vary depending on the type of cells and tissues.Beotime's Ad-mCherry-p62 employs a mature E1-deficient recombinant adenovirus vector system which can only amplify in E1 expressing cell lines such as HEK293A and HEK293, but not in ordinary cells, thus effectively reducing the risks of this product.p62, also known as sequestosome I (SQSTM1), is highly conserved in multicellular organisms (excluding plants and fungi). It is predominantly distributed in cytoplasm, but also present in nuclei, autophagosomes, and lysosomes. p62, a stress-induced protein, plays an important role as a signaling hub in many cellular events such as amino acid sensing and oxidative stress, and also serves as a scaffold protein for PKC, ERK1, mTORC1, NF-кB, and caspase-8, among others. p62 is 440 amino acids long, with multiple domains such as a PB1 domain, a zinc finger domain, two nuclear localization signals, a TRAF6 binding domain, and a LIR domain. The N-terminal PB1 domain is required for oligomerization of p62 to form protein aggregates especially when cells are exposed to oxidative environments, and acts as a signaling organization center responsible for recruiting ubiquitination-modified protein substrates. The LIR domain contains a cluster rich in acidic and hydrophobic amino acids (DDD and WxxL). The acidic cluster and the other two key amino acids of the LIR domain, Trp338/Leu341, interact with LC3 protein at multiple sites, which is responsible for directing the degradation of ubiquitinated proteins into the proteasome or autophagosome. The interaction between p62 and LC3 is also necessary for the autophagic degradation of p62 itself, and inhibition of autophagy will lead to a large accumulation of p62, followed by the formation of p62 and ubiquitin staining-positive aggregates. Caspase-8 is reported to interact with p62 to initiate non-death receptor-dependent apoptotic signaling pathways.Autophagy is a highly evolutionarily conserved intracellular metabolic pathway for partial degradation of its own components through lysosomal phagocytosis, which is associated with a variety of physiological functions. Under unfavorable environmental conditions such as starvation, cells degrade excess or abnormal intracellular components through autophagy to provide energy and raw materials for cell survival and to promote growth and development, cell differentiation, and response to environmental changes. Abnormalities in autophagy are closely related to a variety of pathological processes such as tumors, neurodegenerative diseases, metabolic diseases, and pathogenic infections. Since cellular autophagy plays important roles in both physiological and pathological processes, it has become a new research topic in the field of cell biology.After infection of cells with the Ad-mCherry-p62, mCherry-p62 usually fluoresces as red puncta or diffusive speckles in cytoplasm and nucleus under fluorescence microscopy. When autophage is inhibited, the red punctas or speckles become larger and more numerous. The fluorescent labeling of p62 is an excellent tool for studying autophagy induction and inhibition, and the clearance of protein aggregates as well.Figure 1. NIH3T3 cells infected with the Ad-mCherry-p62 were stained by Hoechst 33342 after 48 hours post-infection. The red punctas or speckles of mCherry-p62 is visible clearly.The titer of this product is ≥1×108 pfu/ml. If cells are infected at 20 MOI, 1ml of this product is sufficient for infecting 10 wells with 500,000 cells/well in 6-well plates, or 50 wells with 100,000 cells/well in 24-well plates. The actual number of wells that can be infected is also dependent on the MOI value used.
Precautions： PrecautionsPrecautions for Adenovirus Use" carefully. This product has a Biosafety Level 1 (BSL-1). It is not known to consistently cause diseases in healthy adults and can be used in accordance 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 while handling the reagent.
Instructions for Use： 1. Determination of the infection conditionsThe following protocol is provided for Ad-mCherry-p62 infection of NIH3T3 cells in 6-well plates. Make appropriately adjustments for the infection of cells cultured in other types of vessels. Different MOI values are required for different types of cells. For first-time users of this product, please optimize the infection conditions. a. Culture NIH3T3 cells in 6-well cell culture plates: The day before infection, seed NIH3T3 cells at 5×105 cells/well in 2ml of complete culture medium. Cells density should be approximately 50% for virus infection. Note: The exact number of cells inoculated is dependent on the cell size and cell growth speed to achieve a cell density of 50% approximately for virus infection next day.b. Calculate the amount of virus required to yield a MOI value of 2, 5, 10, 20 and 40, respectively. Please refer to Appendix 2 for the calculation.c. Thaw the virus on ice and mix well before use.d. Take out the 6-well plate from the cell culture incubator and examine cells by microscopy to make sure cells are in good growth. Replace the old culture medium with 1.2ml of fresh culture medium per well, and add an appropriate amount of Ad-mCherry-p62 to yield a specific MOI value per well. At the meantime, set up wells without virus as negative control. Note: Fresh culture medium should be added to each well as little as possible to increase the efficiency of virus infection. For multi-well plates with a small volume of culture medium, such as 96-well and 48-well plates, the Ad-mCherry-p62 can also be diluted with culture medium to the desired MOI value before addition.e. After 24 hours post-infection, remove the culture medium containing virus. Add 2ml of fresh complete culture medium per well, and continue to incubate for 24 hours before examination by a fluorescence microscopy (Figure 2). The optimal MOI value is the one that provides strong fluorescence with high infection efficiency and has no significant influence on cell growth.Note 1: Higher infection efficiency is not necessarily the better, because too high infection efficiency may easily cause cytotoxicity and interfere with the detection of autophagy. An optimal infection efficiency is usually 20-70%.Note 2: The expression of fluorescent mCherry-p62 can be detected usually 24 hours post-infection, and peaks after 48 hours approximately.Note 3: In cases of high cytotoxicity after adenovirus infection, replacing with fresh complete culture medium 6-12 hours after infection can be attempted.Figure 2. NIH3T3 cells infected with Ad-mCherry-p62 (Beytome, #C3016) at different MOI values as indicated in the figure. Cells were examined 48 hours after infection. mCherry-p62 has an excitation maximum at 587nm and an emission maximum at 610nm.2. Cell infection, autophagy inhibition and fluorescence observationa. Perform infection with Ad-mCherry-p62 at the optimal MOI value determined in step 1. b. Induce autophagy with EBSS (Beytome, #C0213/C0214). Cells without EBSS treatment are used as negative control.c. Inhibit autophagy with autophagy inhibitors such as chloroquine or bafilomycin A1.d. Examine the changes of red fluorescent mCherry-p62 by fluorescence microscopy.Appendix：1. Biosafety practices for use of adenovirusa. As a relatively safe virus, the adenovirus genome does not integrate into the host genome or replicate inside cell after infection, but its potential biological risks can not be excluded. We recommend users reading this specification carefully before handing virus and following it strictly during the operation. For more information about the US CDC biosafety levels and their handling and protection requirements, please visit the website at:http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_sect_IV.pdf.b. Use appropriate biosafety cabinets when handling the adenovirus, which may vary for different adenoviruses. If a laminar flow hood is used, please turn off the blower fan to prevent the contaminated dust with virus from being inhaled. c. Please wear disposable hat, mask, gloves and lab coat to avoid direct contact with the adenovirus. Do not handle the virus when there are wounds in open part your body.d. When handling the virus, be careful not to produce aerosol or splash. If there is virus contamination in the laminar flow hood or on other utensils, wipe it off immediately with 70% ethanol or 2% SDS solution, or take other appropriate measures.e. If centrifugation is required, seal the centrifuge tube tightly before centrifuge. A dedicated centrifuge for virus operation is preferred.f. To examine cells after infection by fluorescence microscope, the following steps should be followed: tighten the culture bottle or plate, clean the outer surface with 70% ethanol, and then examine cells by microscopy. After examination, clean the microscope stage with 70% ethanol.g. All virus contaminated tips, centrifuge tubes, culture vessels, cultures, gloves should be sterilized overnight in disinfectant or 2% SDS solution before discarding.h. Wash hands with soap or sanitizer after removing gloves.2. Calculation of viral MOIMOI (Multiplicity of Infection): A ratio of the number of viruses to the number of cells to be infected.Pfu (Plaque forming units): The number of biologically active virus particles.pfu can be calculated according to the following formula:pfu required = number of cells x MOIFor example, to infect 1×105 cells with a virus at a MOI value of 2, the required pfu is 2×105. If the titer of viral stock is 1×108 pfu/ml, then (2×105 pfu)/(1×108 pfu/ml) = 0.002ml of viral stock should be used.3. Table 1. Summary of Recommended Biosafety Levels for Infectious AgentsBSLAgentsPracticesPrimary 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 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.
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