FACS Perameters for the Secretion Assay

Using the GMD secretion assay, protein(s) secreted by individual cells can be quantified and individual cells of interest can be isolated and recovered using FACS. The ability to sort live cells  of interest based on amount of protein secretion for further culture and study is not possible using other methods.

 

The panel on the left depicts a forward versus side scatter dot plot generated on an EPICS Elite™ (Coulter Corp., Miami, FL) equipped  with a 100 µm orifice quartz flow cell. A linear forward scatter and a log side scatter gain are used respectively to resolve singly occupied from unoccupied and multiply occupied GMDs. Unoocupied GMDs are then excluded from acquisition by increasing the forward scatter threshold or discriminator until they no longer appear on the screen. 10,000 occupied GMD events are collected and a tight gate is set around the singly occupied GMDs for data analysis and sorting.

The panel on the right depicts a forward scatter versus propidium iodide fluorescence intensity  dot plot. A linear forward scatter and a log  PI fluorescence intensity gain were used respectively to resolve occupied GMDs containing live cells from those containing dead cells. A gate is set around the occupied GMDs containing live cells for data acquisition and sorting.

 

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