Cls3770

Adipogenesis was assessed by staining with Bodipy 493/503 (Invitrogen catalog no. D3922) for lipid accumulation and Hoechst 33342 (Thermo Fisher catalog no. 62249) for nuclei³² . Briefly, cells were differentiated in 384 well tissue culture plates (Sigma-Aldrich catalog no. CLS3770), fixed with 4% paraformaldehyde, stained, and imaged on a Keyence inverted microscope (BZX-710) using Keyence BZ-X Viewer Software (1.3.1.1) with the following filters: DAPI (ex, 360/40 nm; em, 460/50 nm; Keyence, OP-87762) and GFP (ex, 470/40 nm; em, 525/50 nm; Keyence, OP-87763) filters. Images were acquired at 20x in a 7×7 tiled grid and stitched to capture the entirety of each well. Tiling and stitching were performed with Keyence BZ-X Analyzer software (1.3.0.3). Image quantification was performed automatically in ImageJ (version 1.52E) using a macro which: 1) Split images into component channels, 2a) for the nuclei channel applied a 3-Sigma Gaussian blur, performed thresholding to identify signal above background, performed watershed to segmentation, and counted the number of nuclei, 2b) for the lipid channel applied a 2-Sigma Gaussian blurm performed thresholding to identify signal above background, and counted the area (#of pixels) with signal above threshold. The amount of adipogenesis was calculated as Lipid Area/#nuclei.