Lambda ls xenon arc lamp light source system

Cultured HEK293-FT cells were trypsinized ≤ 2 min, counted, and 75,000 cells were seeded onto uncoated 18 mm glass coverslips. After 24 h growth, cells were transfected using OPTI-MEM serum free medium, X-tremeGene 9 (Sigma, 6365779001) and various plasmids. Living cells were imaged in a Tyrode’s salt solution (in mM: 135 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 25 HEPES, 10 glucose, pH 7.4) at RT 24–48 h post-transfection. An Observer.Z1 microscope (Zeiss) with a 63x plan-apochromat, 1.4 NA oil objective (Zeiss), Lambda LS Xenon Arc Lamp Light Source System (Sutter Instruments), AxioCam MRm camera (Zeiss), and Zen Blue software (Zeiss) were used for image acquisition. Three-filter FRET images were captured using appropriate filter cubes (Semrock) housed in the microscope turret. CFP cube: (Ex. 438/24 nm, Em. 483/32 nm, Di. 458 nm), YFP cube: (Ex. 500/24 nm, Em. 542/27 nm, Di. 520 nm), and FRET cube: (Ex. 438/24 nm, Em. 542/27 nm, Di. 458 nm). ImageJ software (NIH) was used for image processing and calculations of sensitized FRET efficiency were adapted from the method of Clemens Kaminski (Kaminski et al., 2014 ) with more details provided below.