I'm going to use gecko library in order to infect primary macrophages and is the very first time I'm approaching this technique, so I have some questions.
I'm following the nat prot paper by Julia Joung (2017) but I have some questions regarding the titration part.
I'm using the 2 vectors system and I will infect macrophages expressing the cas9.
I red in the paper that infecting a very low MOI is a very crucial step, to allow just one guide (or so) to enter one cell.
My question is regarding how to calculate the MOI: in my system I don't have any reporter, such as GFP or other molecule to track, so I just want to ask if it is correct to do the titration as follow:
- i will transfect Hek293T cells with Pax, VSVG and the library. i will use petri dish, so I scale up to use 16 petri (instead of 4 t225) and 122.4ug from the library.
- after 8hours I will remove the supernatant and replace with fresh medium.
- after 2 days from the starting point, i will collect the supernatant and freeze at -80
at this point i will thaw it to infect my primary macrophages and calculate the titer as follow:
- day 0: seed macrophages at 1x10^5 per well in 24 well plate
- day 1: add to 1ml of medium the virus in 100ul. Make 1:10 dilution: 1:1, 1:10, 1: 100, 1:1000. keep 1 non-infected well
- day 2 add puromycin and leave until the cells in non-infected well result dead
- calulate the titer as: titer: cn/ vv/ mD where cn: number of cells infected at day of infection; vv: volume of virus used for 1:1 infection in ml (100ul) and mD: dilution at last well that showed 100% of infected cells (counted).
Is this method correct?
Final question is: I will use gecko library that contains around 130.000 sgRNAs. I'm planning to infect around 2*10^8 macrophages. Is this a good number?