Phenoplate 96 well

HEK293 cells were plated at a density of 10,000 cells per well into tissue culture-treated clear bottomed, optical microscopy quality, 96 well plates (Perkin Elmer, PhenoPlate 96-well) and grown for 1 day to ~40–60% confluency. Twenty-four hours after plating, cells were transfected with 50 ng of pcDNA3.1(+) plasmid encoding a N-terminally tagged CRHR2α construct using Lipofectamine 3000 (Thermofisher) following manufacturing protocols. Twenty-four hours following transfection, agonists were then added for 30 min and cells were incubated at 37 °C. Prior to imaging, cells were washed once with PBS and fixed with ice-cold 4% paraformaldehyde in PBS for 15 min. After washing 3 times with PBS, cells were kept in the Alexa Fluor 647 anti-HA (1:2000 dilution, BioLegend cat# 682404) diluted in 5% normal goat serum in PBS. Two hours following antibody incubation at room temperature, the cells were washed three times in PBS and the nuclear stain Hoeschst 33342 (10ug/ml in PBS) and the acidic compartment stain, LysoTracker Deep Red (Thermofisher) was added for 30 min prior to imaging. A LSM880 Airyscan on a Carl Zeiss microscopy system was used for all image acquisition. Confocal laser excitation lines of 405 nm, 488 nm, and 633 nm were used for DAPI, Alexa fluor 488 (FAM), and Alexa fluor 647 fluorochromes, respectively using a Plan-Apochromat 63x/1.0 objective.

PhenoPlate 96-well (PerkinElmer) previously coated with geltrex was used for immunocytochemistry. Cells were fixed in 4% paraformaldehyde for 15 min at RT. Then, cells were washed three times with PBS. For permeabilization, cells were incubated 1 h at RT in PBS, 0.4% Triton-X, 10% goat serum and 2% BSA. After 1 h, cells were washed twice with PBS. Primary antibody was diluted in PBS, 0.1% Triton-X, 1% goat serum and 0.2% BSA. Cells were incubated with primary antibody shaking overnight at 4 °C. Next day, cells were washed three times with PBS. Then, secondary antibody was diluted in the same buffer as the primary and incubated for 2–3 h at RT. Finally, cells were washed three times with PBS. In the first wash, DAPI was added to the PBS (5 µg/ml) and incubated for 15 min at RT. Images were taken using a Zeiss spinning disk confocal microscope. Image processing was done in ImageJ.