Here we present the strategies used to define the cellular expression of a weakly expressed immune gene tagged to tdTomato (Tom) fluorescent protein reporter in murine lung subsets carrying varying levels of autofluorescence (AF). WT and Tom+/+ samples were analysed by the conventional CytoFLEX LX and spectral Aurora 5L cytometers. A broad antibody panel for tissue resident populations was designed to accommodate both cytometers whilst minimizing spectral spillover and compensation/ unmixing-derived spread into Tom.
CytoFLEX, with exquisite sensitivity for Tom emission, revealed expression within 3 subsets including abT cells (low AF) and alveolar macrophages (AMF; high AF). Interestingly, the % of Tom+ in the high AF AMF (but not low AF abT cells) varied greatly depending on the 2D plot partner detector, with the best results observed against V610 (AF correlating). Similar distribution, yet marginal resolution was found by Aurora Spectroflo. For a fair comparison, Aurora raw data was re-unmixed via FCS Express 7 (that preserves raw parameters after unmixing), revealing Tom+ frequencies matching CytoFLEX on 2D plots vs V9 raw detector.
Next, WT samples were spectrally unmixed (U) and subsets evaluated for AF levels at the Tom channel, revealing a 10-100-fold MFI difference between AMF and abT. Based on our previous data we extrapolated that unmixing with AF extraction (U-AF) would result in a substantial increase in signal resolution in the high AF AMF. To this effect, pure AMF spectrum from unstained WT samples was used as a reference tag to extract the AF component from labelled AMF. The resulting background fluorescence of the AMF population resembled low AF cells, increasing the % of AMF Tom+ cells compared to U alone. AMF U-AF also ‘corrected’ the intensity of other panel antigens.
Our data demonstrate critical factors in discovery projects involving rare and dimly expressed fluorescence impacted by high AF.