Brunello Virus Titration

I have a couple of questions: We decided to simply purchase concentrated lentiviral preparations of the Brunello 1 vector library (Addgene cat. no. 73179-LVC).

1. Since this is extra concentrated (1.3×108 TU, titer ≥ 5×107 TU/mL), when I perform titration to determine MOI, should I start at a much lower volume of virus--(10 uL or perhaps even 1 uL)?

2. Is there an advantage to calculating MOI with the cell titer glo assay versus simply counting cells?

3. What is the best concentration for polybrene? It seems most people use 8 ug/mL, but Doench et al. Nat. Biotechnology 2016 says it is 1 ug/mL.

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Fuchsia Kelpieover 4 years ago

1. Yes start with a much lower volume of the virus. Do you know how many times the virus was concentrated? If so, you can scale the titer linearly to start.

2. Cell titer glo assay is much less work than counting cells, and it also samples a larger number of cells, so I switched from counting to cell titer glo.

3. I've been using 8ug/mL, and hasn't been an issue with so I never tried other concentrations.

Mauve Dragonover 4 years ago

Just a question in regards to the cell titre glo assay - I have been using crystal violet instead because that's what we have at the lab and it's the assay I've been using for all my other viability assays and IC50 calculations. I know it's slightly less accurate than an ATP-based assay for example, but do you think that would make too much of a difference to the MOI calculations?

Fuchsia Kelpieover 4 years ago

I think it sort of depends on how much less accurate the crystal violet is. I can't say for sure whether it can be a replacement for cell titer glo, but if you are concerned about accuracy you can always shoot for an MOI of 0.25 for your screen.

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