High-content siRNA 3D co-cultures to identify myoepithelial cell-derived breast cancer suppressor proteins

摘要

Workflow for high content 3D co-culture. SMARTpool siRNA transfection (a) Cells were cultured to ~70–80% confluence, trypsinised and plated at 6000 cells/well in a 96 well plate. Cells were reverse transfected in duplicate using the ALH300 robot at 40 nM final concentration of targets and controls using DharmaFECT 3. Using a BioTek406 liquid handling workstation, media was changed after 24 hours. (b) 48 hours post transfection, 1 plate of cells was stained with Hoechst and imaged using the CX7 or ArrayScan. Images were then run through inForm software to count nuclei to give an overall cell number per well. (c) At 48 hours post transfection the duplicate plate was trypsinised and contents of well transferred to a 96 well round bottom plate with 6000 MDA-MB-231 cells per well. Cultrex was laid in 48 well plates followed by seeding of N1ME & MDA-MB-231 cells from the 96 well round bottom plate. Media was changed on D4. (d) 3D cultures were stained with Hoechst and imaged using the ArrayScan. Tiled images were then run through an Image J script to determine the perimeter of each individual colony and the convex hull. This information generated the perimeter to convex hull ratio for each individual colony which is graphed to give the overall average ‘roundness’ of the 3D co-culture. Created with http://BioRender.com.