Lenti-CMV-EGFP-p65-EF1α-Puro

Item Number

L747625

• Specifications & Purity: 10^9TU/ml
• Stability And Storage: Store at -80℃ for up to 1 year, -20℃ for up to 1-2 months, or 4℃ for up to 1 week.
• Storage Temp: Store at -80°C
• Shipped In: Ice chest + Ice pads

Product Description

Lenti-CMV-EGFP-p65-EF1α-Puro, i.e., Lentivirus expressing EGFP-p65 fusion protein and puromycin, is a recombinant lentivirus developed by aladdin, which can be used in most mammalian cells (including primary cells and stem cells) to express the EGFP-p65 fusion protein driven by the CMV constitutive promoter. The stable infected cells can be selected with puromycin. This product is mainly used to detect the activation and inhibition of NF-κB signaling pathway, and to observe the nuclear translocation of NF-κB by fluorescence microscopy, etc.NF-κB (Nuclear factor kappa-light-chain-enhancer of activated B cells) is an important nuclear transcription factor involved in cellular response to external stimuli, inflammatory response, immune response and other processes, and is present in almost all types of animal cells. NF-κΒ consists of five subunits

Precautions：

Repeated freezing and thawing will reduce the viral titer. If necessary, please store in aliquots after receiving this product. Aliquots must be performed on ice. This product can be stored at 4ºC if it is used within a week, but it should be noted that the viral titer decreases over the time of storage at 4ºC. If stored at -80ºC for more than one year, the titer may decrease, and it is recommended to re-titrate this product.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 during virus operation, 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 the virus should be disinfected by soaking in 1% SDS solution or disinfectant for more than 30 minutes, or sterilized by autoclaving at 121ºC 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 conditions:MOI (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, bu 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 plate.For cells cultured in other type 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. The cell density should be about 70-80% 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 are needed for infecting 1 × 105 HEK293T cells, the required TU is 2 × 106TU (1 × 105 × 20)。For a virus stock with a titer of 1×109 TU/ml, the volume of the virus stock 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 literaturec.Set up the 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 infection. 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 requirements of users.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 cells. Note: When the needed amount of virus stock solution is too small, dilute the virus stock solution appropriately with culture medium before adding to platese.At 4 hours postinfection (hpi), add 250μl of fresh culture medium to each well.f.At 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 should be replaced at least 4 hours after the virus is added.g.After culturing for another 48-96 hours, check the cell growth status and EGFP expression. The optimal MOI value and post-infection time are those under which cell growth is relatively good, with strong EGFP expression and high infection efficiency to facilitate the examination of EGFP.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:TThe infection efficiency is not necessarily the higher the better for luciferase assay, because too high of infection efficiency could cause cytotoxicity. The infection efficiency of 20-70% is generally sufficient.Note 2: EGFP expression can be examined after 72 hours of lentivirus infection, and there is usually a stronger EGFP expression at about 96 hpi.Note 3: In the case of strong cytotoxicity after infection with lentivirus, replacing the virus-containing medium with fresh complete culture medium at 4 hpi can be attempted.2.Infection of cells and fluorescence observation:Perform the virus infection as described in step 1. After the green fluorescence of EGFP-p65 is visible, or after obtaining the cell lines stably expressing the EGFP-p65, subsequent experiments can be performed. Appendix: 1.Safety specifications for using of 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: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.References:1.Liu, T., Zhang, L., Joo, D. et al. Sig Transduct Target Ther. 2017. 2:e17023.2.Giridharan S, Srinivasan M. J Inflamm Res. 2018. 11:407-419.Related Products: Cat. 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