**Total internal fluorescence (TIRF) microscopy of phosphoinositide reporters**
TIRF microscopy which allows selective imaging of the small cell volume, including the plasma membrane, directly adjacent to the coverslip onto whih cells have been seeded. HeLa or A549 cells were seeded in Matrigel-coated (Corning #354230; diluted in Opti-MEM at 1:50) 8-well chamber slides (glass bottom, 1.55 refractive index; Thermo Fisher Scientific #155409) at a density of 5,000 cells/well. The following day, cells were transfected with 50 ng (A549) or 10 ng (HeLa) PIP3 reporter plasmid (GFP-PH-ARNOI303Ex2)51 using FuGENE® HD Transfection Reagent (Promega #E2311), at a 3:1 Fugene:DNA ratio according to the manufacturer’s instructions. To ensure low yet uniform expression of the reporters in HeLa cells, and to aid in the identification of the critical TIRF angle for imaging, these cells were also co-transfected with 200 ng iRFP-tagged Paxillin plasmid (generated by conventional restriction enzyme-based subcloning from an mCherry-Paxillin plasmid, Addgene #50526). In separate experiments, HeLa cells were also transfected with 10 ng or 50 ng of the PI(3,4)P2 reporter mCherry-cPH-TAPP1x3; the use of 50 ng of this reporter enabled easier visualisation in the TIRF field, however the kinetics of the response remained unchanged and results from both experiments were pooled.
Following another 24 h post-transfection, cells were switched to 150 µl serum-free Fluorobrite™ DMEM (Thermo Fisher Scientific #A1896701; supplemented with L-glutamine (2 mM) and 1% penicillin-streptomycin for 3 h prior to time-lapse imaging on a 3i Spinning Disk Confocal microscope fitted with a sCMOS Prime95B (Photometric) sensor for TIRF, with full temperature (37°C) and CO2 (5%) control throughout the acquisitions. A 100X 1.45 NA plan-apochromatic oil-immersion TIRF objective was used to deliver the laser illumination beam (40-50% power) at the critical angle for TIRF and for acquisition of the images by epifluorescence (300-500 msec exposure) using single bandpass filters (445/20 nm and 525/30 nm). Acquisition was performed in sequential mode, without binning, using Slidebook 6.0 and an acquisition rate of 2 or 3 min as indicated. Individual treatments were added at the specified times at 2x to 5x concentration in the same imaging medium, ensuring correct final concentration and sufficient mixing with the existing medium solution. BYL719 (Advanced ChemBlocks Inc #R16000) was used at at a concentration of 0.5 µM.
Image analyses of total reporter intensities were performed with the Fiji open source image analysis package99. The region of interest (ROI) corresponding to the footprint of the individual cell across time points were defined using minimal intensity projection to select only pixels present across all time points, following prior subtraction of camera noise (rolling ball method, radius = 500 pixels) and xy drift correction, intensity levels over time were measured. These analyses were performed with a custom-written FIJI/ImageJ macro. A second macro was used to generate scaled images, with normalisation of all pixels to pre-treatment average intensity (Ft/Fbaseline). All other quantifications were performed using the open source software R/RStudio.
All raw and processed data, including images and analyses scripts, are provided in the associated components. Each can be downloaded as a separate zip file and the scripts run in R provided that the current folder structure is retained. For questions, please contact Dr Ralitsa Madsen (r.madsen@ucl.ac.uk).
