Andor ixon3 emccd camera

Hela cells were transfected with D1ER and PRMT1 construct or siRNA against PRMT1. After 2 days, cells were stained for 10 min with 200 nM MitoTracker Red CMXRos and imaged directly with 488 nm laser excitation (D1ER) and a 561 nm laser (MitoTracker Red CMXRos) with a CFI SR Apochromat TIRF × 100 oil (NA1.49) objective mounted on a Nikon-Structured Illumination Microscopy (SIM) System equipped with an Andor iXon3 EMCCD camera. Three-dimensional (3D) SIM was used in both channels with 30 ms exposure time per image. SIM images where reconstructed using Nis-Elements (Nikon). Images were background corrected with an ImageJ-Plugin (Mosaic Suite, background substractor). Further, the ImageJ/Fiji tool coloc2 was used to determine Pearson and Mander's coefficients, whereby images were thresholded with Costes automatic threshold to determine Mander's coefficient (overlap of mitochondria with ER).

RAW264.7 macrophage cells were seeded on glass coverslips at $2.0\times {10}^5$ cells/ml in complete medium for 24 h and then activated with LPS and IFN-γ following the procedure described above. For all single-phagosome pH studies, the recognition of pH-Sensors (i.e., pH-RotSensor and pH-MagSensor) by macrophages was synchronized following a previously reported protocol^{77 (link)}. Briefly, cell samples were cooled on ice for 3 min to postpone phagocytosis. pH-Sensors were then added at a particle-to-cell ratio of ~5:1. Bindings of particles on macrophage were synchronized by centrifuging cell samples at 200 × g for 30 s. Live cell imaging was conducted in Ringer’s solution at 37 °C on a Nikon Eclipse-Ti inverted microscope equipped with a 1.49 N.A. ×100 TIRF objective (Nikon, Tokyo, Japan) and an Andor iXon3 EMCCD camera (Andor Technology, Belfast, U.K.). Fluorescence emissions at three wavelengths (ex: 488, 561, and 660 nm; em: 515, 586, and 680 nm) were acquired for the time-lapse imaging. The acquisition rate was 2 s/frame. Single-phagosome pH assay in actin-GFP expressing RAW264.7 macrophages was conducted following the same procedure described above.