Flow Cytometry Analysis of GFP Expression

Cells were trypsinised and 200 μl of the suspension was added to a round bottom 96-well-plate for analysis in a BD FACSArray™ Bioanalyzer. GFP fluorescence was excited with a 488 nm argon laser. During analysis of cytometry plots, live cell populations were gated by plotting forward-light-scatter versus side-scatter to visualise and isolate the viable population. GFP-positive populations were determined by plotting the emission from the green channel (detected using 530/30 nm band pass filter) against emission from the yellow channel (detected using 575/26 band pass filter), to compensate for auto-fluorescence events. Unless mentioned otherwise, non-transduced populations were used to set the baseline for GFP expression.
During NIGW investigations, homozygous 3′NIGW vectors (without a recognised promoter) expressed low-level GFP, which was presumably driven by an IRES-mediated promoter trap^{51 (link), 52 (link)}. For this reason, the baseline for GFP was gated against a homozygous control (5′wtDIS + 3′wtDIS sample) to compensate for any IRES-driven expression from unrecombined proviruses. The gated GFP-positive cell populations were used to estimate the amount of reconstituted, full-length proviruses driven by the SFFV promoter.
Where mentioned, GFP-positive cells were sorted on a MoFlo sorting machine.
All FACS data were analysed by FlowJo software version 9.3.1 (©Tree Star, Inc).