Lambda dg 4

TIRF was carried out on Olympus IX81 microscope equipped with Lambda DG-4 (Sutter Instruments, Novato CA) widefield illumination system and Evolve 512 EMCCD (Photometrics, Tucson, AZ) camera, with Cell-TIRFTM (Olympus) illuminator and ×60/1.45NA oil objective using Slidebook 6.0.15 (Intelligent Imaging Innovations, Inc. Denver, CO). The cells being imaged were maintained at 37 °C in the Tokai Hit microscope stage top ZILCS incubator (Tokai Hit Co., Japan). For TIRF imaging laser angle was set to obtain penetration depth of 70–120 nm.

The intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured as previously described. Briefly, [Ca²⁺]_i was measured using the Lambda DG-4 fluorescence measurement system (Sutter Instruments, Novato, CA, USA). Cells were placed on glass coverslips and loaded with Fura-2/AM in darkness for 30 min at 37 °C. After dye loading, the cells were washed and transferred to a perfusion chamber on a fluorescence microscope. Fura-2 signals were obtained by alternating excitation at 340 or 380 nm, and detecting emission at 510 nm. A normal physiological salt solution was used for bath perfusion that contained 135 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 5 mM HEPES, and 10 mM glucose (pH 7.4). The data were analyzed using MetaFluor software (Sutter Instruments). All [Ca²⁺]_i measurements were performed at 37 °C using a heat controller (Warner Instruments, Hamden, CT, USA).

Cultured BNL1 ME cells were perfused continuously at 2 ml per min with DMEM and examined with an inverted microscope equipped with Epi-Fl attachment, perfect focus system (Nikon) and EXi Aqua monochromator (QImaging). Doxorubicin fluorescence was measured as absorbance at 480 nm (580 nm for emission, Lambda DG4, Sutter Instruments). Images were taken every 10 min, monitored online, and analyzed offline using Nikon Elements AR Software (Nikon). 2-APB (200 μM) or CBD (10 μM) and doxorubicin (1 and 5 µM) were bath applied using a fast-step valve control perfusion system, at a rate of ∼1 ml per min.

Cortical neurons were cultured on coverslips and were loaded in Tyrode’s solution with 5 μM Fura-2 AM (Abcam) and 0.02% Pluronic F-127 (Invitrogen) in incubator for 30 min. Neurons were pre-incubated in 5 mM [K⁺]_o solution and perfused with high [K⁺]_o solution, then washed out by 5 mM [K⁺]_o. Fluorescence ratio (F₃₄₀/F₃₈₀) was achieved with a standard ratiometric Ca²⁺ imaging system, which included an inverted IX71epi-fluorescence microscope (Olympus), a camera of EMCCD DU-897D (Andor Technology), the light source of Lambda DG-4 (Sutter instrument), the excitation filters of 340 nm and 380 nm: FF01-340/26-25 (Semrock) and FF01-380/14-25 (Semrock), the emission filters of ~510 nm: FF01-504/12-25 (Semrock), and the dichromatic mirror: DM 400 in U-MWU2 cube (Olympus). Images were acquired and analyzed by MetaFluor software (Molecular Devices).

The [Ca²⁺]_c was measured according to our previous publication [36 (link)]. Briefly, neuron-plated cover slips were exposed to Fura-2-AM (5 μM) for 30 min at room temperature, washed 3 times with regular Tyrode’s solution, and given 30 min for de-esterification. For Ca²⁺ microfluorometry, the fluorophore was excited alternately with 340-nm and 380-nm wavelength illumination (150W Xenon, Lambda DG-4, Sutter, Novato, CA), and images were acquired at 510 nm using a cooled 12-bit digital camera (Coolsnap fx, Photometrics, Tucson, AZ) and inverted microscope (Diaphot 200, Nikon Instruments, Melville, NY). Recordings from each neuron were obtained as separate regions (MetaFluor, Molecular Devices, Downingtown, PA) at a rate of 3 Hz. After background subtraction, the fluorescence ratio R for individual neurons was determined as the intensity of emission during 340-nm excitation (I₃₄₀) divided by I₃₈₀, on a pixel-by-pixel basis. The [Ca²⁺]_c was then estimated by the formula K_d·β·(R–R_min)/(R_max−R) where β = (I_380max)/(I_380min). Traces were analyzed using Axograph X 1.1 (Axograph Scientific, Sydney, Australia). Activation-induced transients were generated by depolarization produced by microperfusion application of KCl 50 mM for 3 s.