I've already written a bit about my troubles regarding the packaging of my lentiviral particles. At the end of my last lentiviral post I had figured out that the packaging of my CherryPicker vector was delayed and optimal collecting time was around 60 hours and not 48 hours. Additionally, I found that the amount of virus-associated p24 (lentivirus capsid) was highest in my target cell medium making harvesting and subsequent transduction quite easy. The problem was, however, that the amount of virus-associated p24 capsid only tells you how much virus you have in your supernatant. It does not indicate the amount of infectious virus in your sample and that's pretty much what you care about when you're harvesting lentiviral supernatant.
Before, when I was using GFP as a reporter gene I would estimate the lentiviral titers by flow cytometry (FACS). I would transduce Huh7 cells in a serial dilution and then analyze the percentage of green cells in a FACS machine and then calculate transduction units per milliliter. Since I don't currently have access to a FACS machine to detect mCherry I thought that I could maybe make use of the TCID50 method to estimate the titer.
I diluted my newly collected lentiviral supernatant into a 96 well plate (similar as shown in the picture) and evaluated what wells still had fluorescent mCherry signal. Then I estimated the titer using the Spearman & Kaerber algorithm. I also estimated the titer of some GFP viruses I had along with my previous CherryPicker stocks so I had something to compare to.
Not only does this method estimate the titer of my lentiviral stocks to be quite similar to what I had already estimated with either p24 ELISA and/or FACS but it also showed that the CherryPicker viruses that I collected after 60 hours had a very high titer. Higher than all of the previous CherryPicker stocks and even the GFP stock.
So it seems that I have solved my lentiviral crisis. At least, this lentiviral crisis. I currently have some other ones I might share later.
Soon I will also post a little bit about my musical guilty pleasure.
Until next time!
Hüldi
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