How is titer of lentivirus determined?

How is titer of lentivirus determined?

The functional lentiviral titer, given in transducing units (TU) per mL, was calculated using the following formula: (1) Where P1 is the percentage of positive (live) cells, N is the number of cells at the time of transduction, D is the dilution factor of the LV used for infection, and the transduction volume V in mL.

How can I improve my lentivirus titer?

found that in some cases, titers can drop 10-fold for every 2kb increase in insert size. To overcome this, lentivirus can be concentrated by a variety of methods such as ultracentrifugation, centrifugation on a sucrose gradient, spin columns, and polyethylene glycol (PEG) precipitation.

What is lentiviral titer?

Lentiviral titer is measured as Transduction Units per ml (TU/ml). One TU produces one integration event in target cells. When the percentage of infected cells is at or below 20%, the number of integrations is approximately equal to the number of transduced cells.

How do you calculate a virus titer from flow cytometry?

The titer of the virus was determined using the following formula: FACS infectious units/ml = [(% of infected cells) × (total number of cells in well) × (dilution factor)]/(volume of inoculum added to cells).

How do you calculate titer Moi?

For example, assuming the titer (functional titer) of the viral particles for a pooled screen is 1 x 10^7 TU/ml (thus 1 x 10^4 TU/µl etc.), for 1 x 10^7 cells, 300 µl of the viral suspension needs to be added to achieve an MOI=0.3.

How do you calculate phage titer?

To calculate the titer (the concentration of phage in the lysate, measured in plaques per ml), multiply back up based on the dilution of the spot. The number of plaques per ml calculated is the titer (concentration of PFU/ml).

How do you store lentiviral particles?

If storing for less than a day, lentivirus can be kept at 4°C. For long-term storage, viral preps should be divided into single-use aliquots, and stored at -80°C. Some reports suggest rapidly freezing virus in a dry ice/ethanol bath or liquid nitrogen prior to storing.

Do you need to concentrate lentivirus?

The routine concentration of the lentivirus requires ultracentrifugation with relative centrifugal force (RCF), typically exceeding 90,000 g8,9,10,11,12,13, to remove impurities and to ensure a high infection rate, especially for in vivo applications.

What are lentiviral particles used for?

These lentiviruses can be used to visualize biological processes as they occur in a wide variety of cell lines, primary cell cultures, stem cells, and non-dividing cells, which may be resistant to standard gene delivery techniques such as transfection or retroviral infection.

What is titer in titration?

In titration, the titer is the ratio of actual to nominal concentration of a titrant, e.g. a titer of 0.5 would require 1/0.5 = 2 times more titrant than nominal. This is to compensate for possible degradation of the titrant solution.

How is lentivirus MOI calculated?

To calculate the amount of virus needed after establishing MOI:

1. (total number of cells per vessel or well) x (desired MOI) = total TU needed.
2. (total TU needed) / (TU/mL functional titer) = total mL of lentiviral particles to add to each well.