So many of my experimental readouts in my scientific career have been fluorescence-based (and for good reason), but especially as we keep doing pseudotyped virus infection assays, it’s becoming really prohibitive to continue reading out infection by flow cytometry (b/c of cost, availability of the instruments, etc), so we’ve recently shifted over to luminescence. As part of this, I wanted to create a recombination vector construct that encodes firefly luciferase, so we can use it as a control when needed. (I also just remembered the other reason we made this plasmid, and it was also to have a luminescence version of testing recombination efficiencies).

Anyway, I recently recombined and selected cells with two independently generated constructs (clones G1402C and G1402D), and determined how many recombined (fLuc-expressing) cells need to be in the well to be detectable. Here’s the resulting plot.

The datapoints on the y-axis/ left edge of the plot are media only, where there is no luciferase enzyme (thus helping to establish the background of the assay). You can tell based on the plot that we start getting detectable values around 10 cells per well, and we’re clearly in the linear range by ~ 100 cells per well. Above that value, it’s clearly in the linear range at least through 250,000 fLuc expressing cells (it’ll be nice to see if we can ever max out the linear range, but that’s probably best done with high MOI pseudotyped virus transductions). The variability between G1402C and G1402D may be error in cell counts, since we know there’s possible (slight) error in those estimates.

Perhaps one day we’ll also start playing around with renilla luciferase and Nanoluc, but for now, we’ll keep playing around with fLuc only just to keep things simple. But ya, now with this plasmid in hand, we can start more comprehensively testing recombination protocols for efficiency without having to book a ton of time at the flow cytometer…