Experiment General FAQs
Q1. How to calculate the Multiplicity of infection (MOI)?I have to do transduction using lentivirus and I have a doubt how to calculate the MOI. I have 5x10 3 ( 5000 viral particles/ ul). How much do I have to take to get MOI of 1 if I plate 10000 cells in a 12 well plate. A : MOI (multiplicity of infection) refers to the number of infected viral particles per cell. It is related to pfu by the following formula: Multiplicity of infection (moi) = Plaque forming units (pfu) of virus used for infection / number of cells.
Your MOI equals 1 means equal number of cells and virus particles and your viral concentration is 5x103 (5000 viral particles/μl) and MOI is 1, so it will take you 2μl virus for 10000 cells.
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. Genemedi got a rich experience in lentivirus production and infection, you could find more information about lentivirus infections on this website: https://www.genemedi.net/i/lentivirus-custom-packaging
Q2. How to calculate properly a lenti/retroviral TITER? I have almost 100% of infected cells (GFP+) by infecting 250000 Jurkat cells by 10 microliters of lentiviral suspension, which were added to a cellular suspension of 250 microliters. By knowing these informations, what should I do to PROPERLY calculate the VIRAL TITER (Viral particles/mL)? A : I think you have added too much virus particles. Here is my protocol of lentivirus titration.
a. Seed 293T 1×10^4 cells/well in a 96-well plate one day in advance.
b. Perform gradient dilution of the lentiviral particles to 1:10, 1:100, 1:1000, 1:10^4, 1:10^5, 1:10^6 in 100ul final volume in culture medium.
c. Total 100ul viral particle mixture should be added to each well with at least 3 replicates per virus.
d. Two days post infection, count the fluorescent positive cells using fluorescent microscopy and select the dilution factor with a proper fluorescent positive proportion (10%-30% positive cells/well). Count the triplicates and average the number of positive cells.
e. Estimate the lentivirus titer using the following formulation: Viral titer (TU/ml) = number of fluorescent positive cells × 10 × dilution.
Genemedi got a rich experience in lentivirus production and titration, you could find more information on https://www.genemedi.net/i/lentivirus-custom-packaging.
Q3. How to calculate the Multiplicity of infection (MOI)? A : I think many researchers have encountered this problem. 8kb gene have exceeded the capacity of most common viral vectors. Effective promoters and expression regulatory elements are essential for gene expression,there is only 6kb gene insertion left for adenovirus, 4kb for lentivirus and 2kb for AAV. However, baculovirus is a good choice for gene expression in mammalian cells, its capacity is very large, even 10kb coding sequence work well in this vector.
Genemedi is experienced in virus production and large gene expression. You could find detail information on this website: https://www.genemedi.net/i/lentivirus-custom-packaging
Q4. How to transfect THP-1 cells? A : We’d like to use lentivirus.
For suspension cells, we recommend using flat fillet centrifuging transfection to infect suspension cells or semi-suspension cells. Add virus suspension into cell culture dish, sealing tightly, and centrifuge at low speed of 200×g for 1 hour in the flat fillet centrifuge. Place cells in cell culture incubator after centrifuging transfection. If the flat fillet centrifuge is inaccessible, you can suspend the cells and transfer cells into centrifuge tubes, followed by low-speed centrifuge, and discard the most of supernatant. Add virus suspension into the tubes, resuspending cells, place it at room temperature for 15 min (no more than 30 min), and transfer the cells and virus suspension into plate to culture. Replace with fresh culture medium the next day. For cells difficult to infect, we recommend repeated infections. Replace with fresh virus suspension 24 hours after the first infection. Repeated infections can increase the infection efficiency markedly.
Genemedi got a rich experience in lentivirus production, titration and transfection, you could find more information on https://www.genemedi.net/i/lentivirus-custom-packaging