Sp2 aobs

Intracellular Ca²⁺ was recorded in single cardiac contracting cells. Single cell preparations were obtained by enzymatic digestion of 7-day-old embryoid bodies for 30 min at 37°C in PBS containing 2 mg/ml Collagenase B (Roche, Mannheim, Germany). Dissociated single cells were plated onto gelatin-coated cover slips in 24-well cell culture plates (Greiner Bio-One GmbH, Frickenhausen, Germany), and cultivated in Iscove's medium supplemented with 15% FCS. Following 24 h of culture, cells were loaded in serum-free medium with 1 μM Fluo-4/AM (Life Technologies) for 30 min. Subsequently, the cover slips were transferred in fresh serum-free cell culture medium to the incubation chamber of a confocal laser scanning microscope (Leica SP2, AOBS, Leica, Bensheim, Germany). Fluorescence excitation was performed at 488 nm, and emission was recorded at 500–550 nm. Sampling rate was 2 frames/s. The fluorescence emission of single cells was assessed by using the image analysis software of the confocal setup.

The TIFF images obtained with the 350 nm (blue), 488 nm (green) and 594 nm (red) wavelength filters of the Leica DM5500B fluorescence microscope were merged using the “Color-Merge Channels” ImageJ function [22] (link). The LEI z-stacks obtained with the confocal microscope Leica SP2 AOBS were merged through Imaris (Bitplane Scientific Software).

Intracellular ROS levels were measured using the fluorescent dye 2′7′‐dichlorodihydrofluorescein diacetate (H₂DCF‐DA) (Life Technologies, Thermo Fisher Scientific), which is a nonpolar compound that is converted into a nonfluorescent polar derivative (H₂DCF) by cellular esterases after incorporation into cells. H₂DCF is membrane impermeable and is rapidly oxidized to the highly fluorescent 2′,7′‐dichlorofluorescein (DCF) in the presence of intracellular ROS. For the experiments, embryoid bodies were incubated in serum‐free medium, and 20 μM H₂DCF‐DA dissolved in dimethyl sulfoxide (DMSO) was added. After 30 min cells were washed in serum‐free medium, incubated for further 10 min, and intracellular DCF fluorescence (corrected for background fluorescence) was evaluated in 3600 μm² regions of interest using an overlay mask. For the assessment of mitochondrial ROS, embryoid bodies were enzymatically dissociated on Day 7 of cell culture, plated onto cover slips and after 48 h stained for 30 min with the fluorescence dye MitoSox Red (8 μM) (Life Technologies, Thermo Fisher Scientific). For fluorescence excitation of DCF, the 488 nm band of the argon ion laser of a confocal laser scanning microscope (Leica SP2 AOBS, Leica) was used. MitoSox Red was excited at 496 nm. Emission was recorded at an emission band of 515–550 nm for DCF fluorescence and >560 nm for MitoSox Red.

HepG2 cells (ATCC HB-8065, Manassas, VA, USA) were cultured and transfected with plasmids encoding GFP-GK and GKRP-mCherry as described previously^{22 (link)}, Fluorescence microscopy of transfected cells was performed as reported earlier^{22 (link)} with a laser confocal microscope Leica SP2 AOBS equipped with a HCX PL APO 63°x/1.4–0.6 Oil Lbd BL objective, and standard filter sets for visualizing 4′,6-diamidino-2-phenylindole (DAPI), GFP and mCherry. Images were processed with the ImageJ 1.48v software (National Institutes of Health, Bethesda, USA).

Synchronized animals were grown until the appropriate stage and then mounted on 2% agarose pads with 10 mM Levamisole to be imaged (10–30 worms in each assay with at least 2 independent experiments). The UPR^ER (Phsp-4:gfp) and vitellogenesis (Pvit-2:vit-2:gfp and Pvit-6:vit-6:gfp) were assessed using an AxioCam MRm camera on a Zeiss ApoTome Microscope. Lipid droplet coverage (Pvha-6:dgat-2:gfp) was analyzed using a Confocal Microscope Leica SP2-AOBS. The UPR^ER (Phsp-4:gfp) on phb-1(tm2751) mutants, the DNJ-27 reporter and ATGL-1 expression (Patgl-1:atgl-1:gfp) was assessed using an ORCA-Flash4.0 LT Hamamatsu digital camera on a Leica M205 Stereoscope equipped with a Plan Apo 5.0x/0.50 LWD objective. Image analyses was performed using the ImageJ software. For the UPR^ER, vitellogenesis and ATGL-1 expression analyses, worms were manually segmented and the mean GFP intensity per worm was calculated. For DNJ-27 reporter the head of the animals was exclude from the analysis. Quantification of the LD intestinal coverage was done in the anterior part of the intestine (int1 and int2 intestinal cells). LDs were segmented in ImageJ and classified as smaller (less than 1 μm²) and larger (equal or bigger that 1 μm²). Data was analyzed using the GraphPad Prism software.