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| 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. |
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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. |
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| 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. |
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