You've harvested your lentiviruses
and are ready to infect your cell line!
But wait- just how many lentiviral particles do you actually have?
In this video, we'll explain the different methods you can use
to measure your viral concentration - called viral titering.
What is titer?
Titer is the measurement of the virus concentration in the prep you harvested.
Knowing the viral titer is important for evaluating the quality of your viral prep
and how much prep is needed for a successful infection.
In this video, we will focus on how to determine lentiviral titer.
We will cover:
Functional vs. Physical titer
How to determine functional titer, including the FACS, Colony Formation Assay, and qPCR-based methods.
How to determine physical titer, including the p24 ELISA and qRT-PCR-based methods.
Which titering method you should use.
How to calculate titer.
Types of Titer
There are two different types of titer measurements: functional and physical titer.
Functional titer measures how many viral particles are actually able to infect cells,
whereas physical titer simply measures how many total viral particles there are
both functional and non-functional.
Functional Titer
The three main methods for determining functional titer are:
1. Counting fluorescent cells using Flow Cytometry or FACS.
2. Counting antibiotic resistant colonies.
3. Counting proviral copies using qPCR.
Let's briefly go through each method.
Method 1 - Count fluorescent cells using Flow Cytometry (FACS).
If your viral construct carries a fluorescent tag or contains an antibiotic resistance gene
you can use the FACS method to determine functional titer.
Note that the FACS method is only suitable for viruses that do not lyse cells such as lentiviruses.
Start by transducing your target cells with serial dilutions of your lentiviral prep.
Then, use FACS to count how many target cells are positive for fluorescence.
This method is especially efficient if your vector carries a fluorescent marker as you won't need to do antibody staining.
Method 2 - Count antibiotic resistant colonies.
Start by transducing target cells with serial dilutions of a viral prep.
Then, treat dilutions with an antibiotic and count how many colony forming units are still present.
Both the FACS and the colony counting method often underestimate viral titer
since both these assays cannot determine if a cell has been infected by more than one viral particle.
Method 3 - Count proviral copies in a cell using qPCR.
Use a qPCR to measure how many proviral copies have been incorporated into the target cell's genome.
This method is quite labor intensive and involves isolating genomic DNA from transduced cells
and generating primers for various viral components.
Physical Titer
Now we will briefly explain physical titer methods
which measure the number of viral particles present regardless of functionality.
The two major methods for determining physical titer are:
1. Measuring p24 expression using ELISA.
2. Measuring viral RNA using qRT-PCR.
Method 1 - Measure p24 expression using ELISA.
You can use the ELISA method to measure virus-specific antigen on the surface of the virus.
For example, use a p24 ELISA kit to determine the amount of the lentiviral capsid protein, p24, in your prep.
A challenge with this method is that you will be measuring all p24, even if it is not incorporated into a lentivirus.
Method 2 - Measure viral RNA using qRT-PCR.
In this method, viral RNA is converted to cDNA.
Then, qPCR primers are used to target specific viral components, allowing viral RNA to be quantified.
abm's qPCR Lentivirus Titration Kit (Cat. No. LV900)
enables quick, one-step quantification of lentivirus in less than 2 hours directly from viral lysate.
The kit comes with unique primers for lentivirus to eliminate NTC amplification and ensure accurate titer measurement.
This kit is calibrated against functional assays
so even though the kit measures physical titer, the values that are calculated are close to results from functional titers.
Which method should you use?
Now that you have an overview of the different titer methods available
which one should you use?
In general, functional titers are more accurate but more time consuming
while physical titers are quick but often overestimated due to inclusion of defective and non-infective particles.
However, for most experiments a measurement of physical titer is satisfactory.
Other Considerations
Viruses can also be sensitive to freeze-thaw cycles
meaning that the titer of a recently collected sample can be much higher
than if the same sample was stored in a freezer and thawed multiple times.
To avoid losing viral titer due to excessive freeze-thaw cycles
it is recommended to aliquot the virus as soon as it is made and store them at -80°C for long term storage.
How to Calculate Titer
Depending on which method you chose, the titer calculation will vary.
In this video, we will focus on calculating physical titer using the qRT-PCR method.
From your qRT-PCR measurements, you will collect cycle threshold (Ct) values from your viral prep.
It is a good idea to also collect Ct values
from positive and negative controls at the same time.
We recommend using at least two standards.
Using these Ct values, calculate titer using the method provided by your kit's vendor.
In abm's kit, the Ct value of the virus samples are calculated
by a logarithmic regression generated by the kit's DNA standard.
Excellent!
Now you have calculated the viral titer of your prep!
Don't rush off to transfect your cells just yet!
You still need to determine how many viral particles are needed
to successfully infect your cells -
a term called Multiplicity of Infection or MOI.
It is important to determine the optimal MOI needed for efficient transduction.
MOI can be affected by factors like inoculum volume, transduction time and the type of target cell used.
We've explained the concept of MOI and how to calculate it in our previous video.
You can refer to that video to learn how to do it yourself!
All done?
Congratulations!
Now that you know your viral titer and MOI
you are ready to transduce your cell line!
Our next video will be on the topic of Lentiviral Transduction.
It will feature our new ViralEntry™ Transduction Enhancer
which boosts transduction efficiency, especially in difficult cell like T cells!
Stay tuned to learn more!
Finally, check out our new abmVacations+ Program!
Earn points with every abm order and exchange them for coffee cards, VISA gift cards and even vacation packages!