My cell culture protocol entails the formation and differentiation of embryoid bodies (EBs) beginning with mouse induced pluripotent stem cells. IPSC are suspended in 20 uL hanging drops to facilitate EB formation. On day two of the protocol, the hanging drops are plated in gelatin-coated 6-well plates containing differentiation medium. The medium is changed daily in order to induce adipogenesis.
Via flow cytometry, I intent to analyze various cell surface markers indicative of cell pluripotency, and mesenchymal and stem-like properties (specifically SSEA1, CD56, and CD133). I have not been able to dissociate the EBs on days 2-6 of the protocol for single-cell analysis. I have tried various cell dissociation protocols, including: accutase, tripsin, TEG, collagenase+cell dissociation buffer, collagenase+accutase, collagenase+TEG. Due to the formation of matrices amongst the cells, strong adherence of EBs to the plates, cell-to-cell adhesion with the EBs, and the degradative effect of collagenase on CD56, specifically, I have not success with any of these suggested methods of EB dissociation. I have read that TryPLE may or may not work.
Please suggest protocols/compounds for EB dissociation!
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