Ixon ultra

Hardware used for the experiments included an Eclipse Ti inverted microscope equipped with a motorized laser TIRF illumination unit, a Borealis beam-conditioning unit (Andor Technology), a 60× Plan Apochromat TIRF 1.49 NA objective (Nikon), an iXon Ultra electron-multiplying charge-coupled device camera, and a laser merge module (LMM5; Spectral Applied Research) equipped with 405-, 488-, 561-, and 640-nm laser lines. All hardware was controlled using Micro-Manager [36 ,38 ] (University of California, San Francisco), and all experiments were performed at 37°C and 5%CO₂.
Activity of opto-PI3K was controlled via a 470-nm (blue) LED (Lightspeed Technologies) that transmitted light through a custom DMD (Andor Technology) at varying intensities by connecting the LEDs to the analog outputs of a digital-to-analogue converter and setting the LED voltages using serial commands via custom Python code. Our microscope is equipped with two stacked dichroic turrets such that samples can be simultaneously illuminated with LEDs using a 488-nm longpass dichroic filter (Chroma Technology) in the upper turret while also placing the appropriate dichroic (Chroma Technology) in the lower turret for TIRF microscopy.

Human 1321N1 astrocytoma cells were loaded with a calcium‐chelating fluorescent dye (Molecular Devices), either fluo‐4 acetoxymethyl ester (fluo‐4 AM for cells transfected with P2X2, P2X4, or P2X7 receptor), or Calcium‐4 AM or Calcium‐5 AM for cells transfected with P2X1 or P2X3, respectively (Baqi et al, 2011).
HEK‐P2X2 sniffer cells were loaded with 2 μM fluo‐4/AM in a humidified incubator for 30 min and washed for 30 min prior to transferring to a perfusion chamber with oxygenated Krebs–Henseleit buffer on the stage of an upright Eclipse FNI Nikon scope equipped with a Andor iXon Ultra high speed camera for real‐time Ca²⁺ imaging. Elements software was used for data acquisition. Ambroxol was dissolved in dimethyl sulfoxide (DMSO) and added to the cells at a final concentration of 20 µM followed by stimulation with ATP at its respective EC₈₀ concentration (P2X1 and P2X3 [100 nM], P2X2 and P2X4 [1 µM], P2X7 [1 mM]). The assay volume was 200 µl and the final DMSO concentration was 1%. ATP activation of the receptors led to increased calcium influx and consequently to increased fluorescence, which is blocked by treatment with antagonists.