Hc pl apo cs2 63x 1.20 water objective

Intracellular free zinc measurement was performed with a slightly modified from a protocol published before [37 (link)]. Briefly, cells were grown in 96-well plate cavities up to 70–75% confluency before loading with 2.5 µM Zinpyr-1 in a loading buffer (10 mM HEPES; pH 7.35; 120 mM NaCl; 5.4 mM KCl; 5 mM glucose; 1.3 mM CaCl₂; 1 mM MgCl₂; 1 mM NaH₂PO₄; 0.3% (w/v) BSA). Next, aerogel swelling supernatans or metal salt solutions (ZnSO₄, ZnO, CaCl₂, AgNO₃) were added and fluorescence was measured after 30 min incubation on a Tecan Infinite M200 reader using excitation/emission wavelengths of 492/527 nm for Zinpyr-1 [38 (link)]. In addition, confocal laser scanning microscopy (Leica TCS SP8 CLMS equipped with LAS X 3.5.5.19976 software platform; Leica, Wetzlar, Germany, using a HC PL APO CS2 63x/1.20 water objective; filters settings were λ_Exc 488 nm/ λ_Em 500–550 nm) was performed to monitor cellular distribution of Zinpyr-1-accessible zinc.

All imaging took place in Leibovitz’s L-15 supplemented with 10 µg/mL cycloheximide (Sigma-Aldrich, C4859) and cells were pulsed with biotin as described above. Imaging was performed on a Leica SP8 SMD system at 37 °C, equipped with an HC PL APO CS2 63x/1.20 Water objective. Fluorophores were excited with a pulsed white-light laser, operating at 80 MHz. mCitrine was excited at 514 nm, two separate HyD detectors were used to collect photons, set at 521–565 nm and 613–668 nm, respectively. Photons were collected for 1 min and lifetime histograms of the donor fluorophore were fitted with monoexponential decay functions convoluted with the microscope instrument response function in Leica LAS X.

For protein localization, wild-type (WT) yeast BY4741 cells were transformed with pUG35 containing either the NgCTR or NfCTR gene, and transformants were grown overnight at 30°C in liquid SC-ura medium, washed and resuspended in phosphate-buffered saline (PBS), pipetted onto a microscope slide and mixed with the same volume of 2% agarose. The microscope slide was then covered with a cover slide and sealed. A fluorescent signal was detected using a Leica TCS SP8 WLL SMD confocal microscope (Leica, Germany) with an HC PL APO CS2 63x/1.20 water objective, excited at 488 nm and detected within 498–551 nm by a HyD SMD detector. The PMT detector was used for bright-field imaging. The resulting images were processed by LAS X imaging software (Leica Microsystems, Germany) and ImageJ (Schneider et al., 2012 (link)). Yeast transformation was performed according to a previously published protocol (Gietz and Schiestl, 2007 (link)).