Imagexpress micro xl high content microscope

Screen and hit validation experiments were performed using the HTS equipment: Echo555 Acoustic transfer system (Labcyte, Germany), Combi MultiDrop (Thermo Fischer Scientific), Washer Dispenser II (GNF, San Diego, CA, USA), EL406 Microplate Washer Dispenser (BioTek, Winooski, VT, USA), Bravo Automated Liquid Handling (Agilent, Santa Clara, CA, USA). Luminescence signals were measured by luminescence module of PheraStar FS plate reader (BMG Labtech, Ortenberg, Germany). Acumen laser scanner (TTP Labtech, Hertfordshire, United Kingdom) and ImagExpress Micro XL high content microscope (Molecular Devices, Sunnyvale, CA, USA) were used to acquire and analyze images.

To induce starvation, Hl5c growth medium was removed and D. discoideum WT, trafE-KO or atg1-KO knock-in act5::ALIX-GFP, act5::GFP-Vps32, act5::GFP-Vps4 and act5::GFP-Atg8a cell lines were washed once with Soerensen buffer (SB, 15 mM KH₂PO_4, 2 mM Na₂HPO₄,, pH 6.0) and incubated for 90 min in SorMC-Sorbitol buffer (15 mM KH₂PO_4, 2 mM Na₂HPO_4, 50 μM MgCl_2, 50 μM CaCl_2, pH 6.0, 120 mM Sorbitol) directly in 96-well black, glass-bottom plates (Perkin Elmer, MA, USA). Images from triplicates with 9 positions per well were recorded using a 60 x water immersion objective with the ImageXpress Micro XL high-content microscope (Molecular Devices). The number of ALIX-GFP, GFP-Vps32, GFP-Vps4 or GFP-Atg8a structures and foci were counted using the MetaXpress software (Molecular Devices, CA, USA), and analysed using GraphPad Prism software.