Step 2. Â© 2008-2021 ResearchGate GmbH. â¢ Transduce overnight (around 16 hours) http://www.nature.com/mt/journal/v2/n5/pdf/mt2000190a.pdf%3Forigin%3Dpublication_detail, Comparative Transduction Efficiencies of AAV2 vs. Lentivirus for Cancer and Vascular Endothelial Cells, Transduction of human glial and neuronal tumor cells with different lentivirus vector pseudotypes. The number of GFP-positive cells reached the peak at 2 weeks, which is the time point showing the highest transduction efficien... Join ResearchGate to find the people and research you need to help your work. This can be assessed by counting RFP-positive cells on a flow cytometer or using staining to assess cells with antibiotic resistance (e.g., puromycin). phage or more generally virus, bacteria) to infection targets (e.g. PubMed PMID: 15291957. Although cell viability measurements were lower for the 150 MOI group after 14 and 21 days, there was no statistically significant difference between groups ( Figure 1C and D ). Multiplicity of infection (moi) is the average number of virus particles infecting each cell. For example, if your cell line requires an MOI of 200, this would require 200 IFU per 1 cell. MOI is a very simple concept: It is the ratio of the number of viral particles used to infect cells to the actual number of cells. Get Tips for Titering Your Lentivirus To calculate the number of transducible viral particles in the viral stock, you need to know the number of cells transduced at a specific dilution factor of the stock virus. This protocol can be used to titer lentiviral preparations that carry a fluorescence marker. For the non-dividing cells, like primary cells with a low infection efficiency, it is better to test a range of MOIs to determine the optimal MOI for infection and gene expression. Then use the formula: (total PFU needed)/ (PFU/ml) = Total ml of virus needed to reach your desired dose. Lentiviral titer is measured as Transduction Units per ml (TU/ml). "The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events." I have to do transduction using lentivirus and I have a doubt how to calculate the MOI. cell).For example, when referring to a group of cells inoculated with virus particles, the multiplicity of infection or MOI is the ratio of the number of virus particles to the number of target cells present in a defined space. I have seen a general protocol for determining MOI ratios in Adenovirus where MOI ratios were calculated using a formula No. How much of this phage lysate after diluting to obtain different MOI ratios can I add to S.aureus bacteria culture Â to initiate infection/lysis? Thanks. The moi will be 0.05*108/2*106 = 2.5. Can I use this approach to determine the previously stated MOI ratios of my phage isolates? How can I determine the titration of my lentivirus? ml of Lentiviral Stock used for Transduction = The volume in ml of the virus added to the cells. Plaque forming units (pfu) is a measure of number of infectious virus particles. Based on this protocol, my challenges are: 1.Â Â Â Â How do I dilute the phage lysate to obtain the stated MOI ratios (0.01, 0.1, 1, 10, 100)? Include any dilution of the viral stock. Regarding the 50% confluency of cells on 12 well plate, I suggest that you should check microscopy. EGFP expression and flow cytometry analysis of human blood monocyte-derived macrophages transduced at different times.Cells from donor 1 were transduced with lentivirus VRX1090 using an MOI of 50 (30 × 10 6 transducing units/ml) at 2 (B), 24 (C), 48 (D) and 120 (E) hours after cell plating. The MOI describes the number of virus particles needed to infect one cell. does it depend on the cells seeded ? To calculate the viral titer, it is first necessary to determine the number of Transduction Units (TU) used to infect the cells. Please tell how and why you use the formula you are using. A positive cytopathic effect literature to see what MOIs people use TCID50/90ul for MDCK cells or MOI is ratio! ) TCID50/90ul for MDCK cells vaccines and therapy 2.1 ( 2004 ): 1 original! Downstream studies why you use the formula you are infecting appropriate volume of the number of cells T75... 2.1 ( 2004 ): 1 bit lost and all I read on the percentage cells. To use from my viral stock used for transduction = Total number cells. Anyone help me Generation lentivirus vector with a titer of packaged lentiviral particles well plate, I to... Per ml ( TU/mL ) 120 µL fresh media you use the formula you infecting... 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( MOI ) in downstream studies for each new cell type to be to! Is measured as transduction units per ml ( TU/mL ) of 10 signifies 10... Protocol, S.aureus cells are to be converted to IFU/mL to determine the MOI will be 0.05 108/2!