A1rsi laser scanning confocal imaging system lscis

Fluorescence imaging experiments were performed with cells seeded at a density of 50,000 cells/plate on Mat-Tek cover-glass plates (Ashland, MA). The plasmid expressing SPLICSs (10.4 nm) was purchased from Addgene (#164108) and cloned into the tetracyclin inducible lentivirus system pLV-Bsd-TRE-CMV-rtTA. AML12 cells were transduced and put under blasticidin selection before being screened for positive clones. Digital images were acquired using a Nikon A1Rsi Laser Scanning Confocal Imaging System (LSCIS; Nikon, Melville, NY) equipped with 405 nm, 488 nm, 561 nm, and 640 nm laser, four-channel GaSP detectors, and a 60x water immersion objective. To determine the subcellular localization of LDs, MAM, and mitochondria under fed and fasted conditions, cells were treated with AutoDOT neutral lipid stand and MitoTracker Deep Red. Media conditions are described above for fed and fasting media. For probe excitation, the A1Rsi LSCIS utilized the 640 nm diode laser (MitoTracker Deep Red), the 405 nm laser line (AutDOT Neutral lipid stain), and the 488 nm laser line (SPLICSs) to acquire images of the cells by sequential excitation. Image files were analyzed using NIS-Elements software or FIJI-Image software. z-stack or multiple focal planes were imaged to ensure compartmentalization and localization.