Description
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 belowPrecautions：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