Bis_104_facs_fall_08 - unbound antibodies Run the double labeled mixed cell suspension through the FACS instrument The instrument will separate the

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Facts about FACS ( F luorescence A ctivated C ell S orting) 1. What is the FACS technique used for? It can be used to obtain pure, homogenous populations of live cells from a mixture of cell types. (For example, Mukouyama used FACS to separate PSN (DRG: dorsal root ganglia) preparations into pure populations of neurons and Schwann cells; it was also used to separate undifferentiated ECs from differentiated ECs.) 2. How does it work? First, identify a cell-type specific marker that is on the surface (PM outer leaflet) that is unique to each cell type you wish to isolate (e.g., IB4 lectin for neurons; p75 LNTR for Schwann cells). Obtain fluor. labeled antibodies that are specific for each cell type marker. Prepare a single cell suspension from the tissue of interest and treat the mixed cell suspension with both antibodies; allow time for recognition and binding, then remove any
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Unformatted text preview: unbound antibodies. Run the double labeled mixed cell suspension through the FACS instrument. The instrument will separate the cell populations based on their fluorescence (e.g., will separate red cells from green cells). 3. What is the end product or result? If the separation works, you would then have 2 separate populations of live, healthy cells that could be used for in vitro experiments. 4. How can the effectiveness of the FACS based separation be verified? By RT-PCR and by direct observation with immunofluorescence microscopy (Mukouyama does this in fig 6D and in fig 7ABCD.). So, what do you think? Did Mukouyama successfully separate neurons from Schwann cells by the FACS technique? What is the experimental evidence that supports the answer you have chosen?...
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This note was uploaded on 01/24/2009 for the course BIS 104 taught by Professor Scholey during the Fall '08 term at UC Davis.

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