Ixon emccd dv887 dcs bv

For imaging, cultured RGCs were plated as a monolayer on 12-mm glass coverslips coated with poly-D-lysine (Sigma). Astrocytes were incubated with 5 µM Fura-2 AM plus 1 µM pluronic acid (Molecular Probes) for 30 min at room temperature. External solution contained 150 mM NaCl, 10 mM HEPES, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 22 mM sucrose, and 10 mM glucose (pH adjusted to 7.4 and osmolarity to 325 mOsm). Intensity images of 510 nm wavelength were taken at 340 nm and 380 nm excitation wavelengths using iXon EMCCD (DV887 DCS-BV, ANDOR technology, UK). Imaging Workbench version 6.0 (Indec System, CA, USA) was used for acquisition of intensity images and conversion to ratios as previously described [37 (link)].

For Ca²⁺ imaging, astrocytes with HEK293T cells transfected with P2X2-V343Q were incubated with 5 µM Fura-2 AM (mixed with 5 µl of 20% Pluronic acid) (Invitrogen, Grand Island, NY, USA) for 30 min and washed at room temperature, and subsequently transferred to a microscope stage for imaging. External solution contained (in mM): 150 NaCl, 10 Hepes, 3 KCl, 2 CaCl₂, 2 MgCl₂, 5.5 glucose, 20 Sucrose, pH adjusted to pH 7.3. Intensity images of 510 nm wavelength were taken at 340 nm and 380 nm excitation wavelengths using either iXon EMCCD (DV887 DCS-BV, ANDOR technology, UK). Two resulting images were used for ratio calculations in Axon Imaging Workbench version 6.2 (Indec System, CA, USA). P2X2-V343Q-mediated currents were recorded from HEK293T cells expressing P2X2-V343Q under voltage clamp (Vh=-70 mV) using Multiclamp 700B amplifier (Molecular Devices), acquired with pClamp 9.2. Recording pipettes were filled with (mM): 110 Cs-Gluconate, 30 CsCl, 0.5 CaCl₂, 10 HEPES, 4 Mg-ATP, 0.3 Na₃-GTP and 10 BAPTA (pH adjusted to 7.3 with CsOH). For simultaneous recording, Imaging Workbench was synchronized with pClamp 9.2.