I would like to perform annexin assay on cultured neurons and I am afraid that accutase incubation, followed by weak centrifugation and resuspension might disrupt neurite membrane. What is the best procedure to keep the neurons intact?
Hello Tatyana, I have used normal trypsin EDTA detachment of neurons (like Neuro2a, IMR32) for many times to perform the FITC-AnnexinV and PI assay during FACS experiments. They worked fine for me. Microscopic examination of those cells showed spherical cells with not much sign of damage interns if membrane rupture or abnormal cell morphology. While using trypsin-EDTA keep it in mind that different neuronal cell lines take different time to get detached. Like neuro2a takes lesser time (2-3 minutes) while IMR32 takes little more time (4-5 minutes) to get dissociated from surface. However, longer incubation of cells with trypsin appears not good for further seeding of cells. Hope this helps.
Thank you very much for the answer. I will try a short incubation time with trypsin and see how it works for my neurons.
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