Iq3 acquisition software

Cells were grown on glass coverslips were stained using standard immunocytochemistry techniques. Briefly, cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100, blocked with 3% BSA and stained with the following antibodies: mouse anti-GM130 (1:250), rabbit anti-TGN46 (1:500), and Phalloidin Alexa Fluor 647 (Thermofisher, A30107). Cover slips were mounted using VectaShield with DAPI (Vector Laboratories, H-1800–2). All images were collected on a Nikon Ti-E inverted microscope (Nikon Instruments, Melville, NY) equipped with a Plan Apo 60× oil objective. Images were acquired using a Zyla 5.5 camera (Andor Technology), using the iQ3 acquisition software (Andor Technology).

HEK293T cells were plated on fibronectin-coated glass coverslips in 12-well plates, at 300,000 cells per well. The following day, cells were subjected to amino acid depletion/or complete media restimulation (see above) and then fixed in 4% paraformaldehyde (in PBS) for 15 min at RT. The coverslips were rinsed twice with PBS and cells were permeabilized with 0.1% (w/v) saponin in PBS for 10 min. After rinsing twice with PBS, the slides were incubated overnight at 4°C with the indicated primary antibody in 5% normal donkey serum, then rinsed with PBS, and incubated with fluorophore-conjugated secondary antibodies produced in goat or donkey (diluted 1:500 in 5% normal donkey serum; Life Technologies) for 45 min at RT in the dark. Coverslips were then washed three times in PBS and mounted on glass slides using VECTASHIELD Antifade Mounting Medium (Vector Laboratories) containing 4′,6-diamidino-2-phenylindole stain. All images were collected on a Nikon Ti-E inverted microscope (Nikon Instruments, Melville, NY) equipped with a Plan Apo 60× oil objective. Images were acquired using a Zyla 5.5 scientific complementary metal-oxide semiconductor camera (Andor Technology), using the iQ3 acquisition software (Andor Technology).