Automation of a Novel Cell-Based ELISA for Cell Signaling Pathway Analysis
Poster Feb 16, 2012
Wendy Goodrich, Peter Banks, Ron Osmond, Antony Sheehan
In addition to offering simplicity, speed, and flexibility, the technology is a highly sensitive technique that lends itself to automation at multiple levels including seeding cells in microplates; cell media exchanges; serial dilution and transfer steps; assay reagent additions; microplate washing; and, signal detection. Assay performance studies were conducted using lysate kit controls to verify optimal automation performance for S/B, Z’, and intra- and inter-assay precision. A ‘one well one day’ cell based assay protocol was developed and used for pharmacology assessment for agonist/antagonist analysis of the PI-3/AKT/mTOR signaling pathway. Procedures demonstrated here are well suited for validating automation of model system protocol development.
We utilized paired synthetic crRNAs coupled with our synthetic tracrRNA in cells transduced with lentiviral Cas9 to perform a functional knockout on hsa-miR-221. This three-part system (crRNA, tracrRNA and Cas9) has demonstrated efficient gene editing when used with only one guide RNA, but the goal was to use two crRNAs to remove the entire stem-loop.READ MORE