Figure 5.

Gating strategy for separating a cell population into subpopulations by flow cytometry based on the transfection of the construct pGlast-dsRed2/pCAG-Venus

(A) Coronal sections of the murine somatosensory cortex of embryonal age E14 after IUE at E12 showing fluorescent protein expression in the target cell population: Venus expression driven by the ubiquitous promoter pCAG (left), dsRed2 expression driven by apical radial glia cell specific promoter Glast (middle), and merged (right). CP: cortical plate, IZ: intermediate zone, SVZ: subventricular zone, VZ: ventricular zone. Scale bar: 50 μm.

(B) Singlets SSC populations of both fluorescence minus one (FMO, single transfected) controls, untransfected, and cotransfected samples with the construct pCAG-Venus/pGlast-dsRed2. The FMO control of the single Venus transfection and the FMO control of the single dsRed2 transfection, together with the untransfected control, were used to calculate the compensation. Compensation with the FMO signals brighter than the signal in the cotransfected sample ensured correct differential fluorescence detection in the cotransfected sample. This compensation and gate setting served as template every time the cotransfected samples were sorted. Note: Based on our experimental question, dsRed2+ cells may also have Venus fluorescence (double positive). This experiment was performed on 20,000 cells of E14 embryonic cortices. At this stage, an additional low false positive signal was visible already in the untransfected condition, which was excluded for sorting.

(C) Computational compensation matrix (“Compensation Wizard” of BD FACS Diva software; recreated with FlowJo) defined values for compensation of spillover into the other bandpass filter.

(D) The bandpass filter (BP) 530/30 covered the emission peak and the spread in the case of FP Venus well, while the BP filter (610/20) covered the emission spectrum of dsRed2 best. The plot was created with FPbase.¹⁷