After performing the GMD Secretion Assay or the GMD Growth Assay, individual cells of interest can be isolated and recovered using FACS. GMD Assays are unique in that they allow the recovery of high numbers of viable cells based on level of protein secretion.

 
 

 
 
Left Panel Above: Identifying Singly Occupied Cells.
The left panel above 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. Linear forward scatter and log side scatter gain are used respectively to resolve singly occupied from unoccupied and multiply occupied GMDs. Unoccupied 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.
 
 


Right Panel Above: Identifying Viable Cells.
The right-hand panel above depicts a forward scatter versus propidium iodide (PI) fluorescence intensity dot plot. Linear forward scatter and 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.

 
 



 
  Simultaneous detection of viable CD3 positive IFNg secreting cells. After stimulating in vitro with antigen in the presence of mitomycin C treated autologous feeder cells and mrIL-2, the GMD IFN gamma secretion assay was performed with 1x106 encapsulated cells. Cells were incubated with FcBlock™ and PE-labeled mouse anti-CD3 Ab (10 µg/ml) prior to flow cytometric analysis. Unoccupied GMDs were discriminated from occupied GMDs using FSC and SSC (panel A). Only GMDs occupied by viable (PI negative) cells were included in data acquisition (R1 gate, panel A). Dot plots of the negative control and the IFNg secretion sample (both stained with anti-IFNg/FITC (FL-1) anti-CD3/PE (FL-2) Abs) are shown in panel B and panel C, respectively.