Indian Institute of Science Education and Research 1 article published in JoVE Cancer Research Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures Abhijith Kuttanamkuzhi1, Libi Anandi1,2, Vaishali Chakravarty1,3, Mayurika Lahiri1 1Biology Department, Indian Institute of Science Education and Research, 2Department of Biology, Center for Genomics and Systems Biology, New York University, 3Enveda Therapeutics India Pvt Ltd. The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.