We're trying to isolate neural stem/progenitor cells from subventricular zones and hippocampi as well as activated mature neurons from olfactorius bulbs of young mice. Debris (especially myelin) is a major obstacle for labelling and sorting of our cells. Is it possible to eliminate debris only by gating FSCa vs SSCa followed by FSCa vs FSCh, or should we use special isolating techniques like percoll, ficoll or MACS?
Any protocol suggestions?
Thank you
Hi Necat, I tried to stain lymphocytes in meningioma samples from patients (brain tissue) and there was a lot of debris which hindered the identification of lymphocytes in the FSC and SSC. However, I tried to load the cell suspension on a Ficoll gradient which considerably improved the purity of the lymphocyte population. The huge mass of debris clearly settled at the bottom with a pure ring of lymphocytes. Though you work with mice, I think ficoll should be very helpful to separate the cells of interest and the debris, however cell numbers may be limiting sometimes.
A gradient centrifugation will certainly help in reducing the debris, especially if you plan to sort next. Don't use MACS, the debris will certainly clog the column. You may also try using the column free bead based isolation from stem cells
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