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Summary of assay conditions that were tested and selected for the autologous tumor IFN-γ ELISPOT assay |
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| Item optimized |
Comparison |
Selected for assay |
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| Plates |
Clear vs. Opaque |
Opaque |
| Targets (tumor cells) |
sCD40Lt stimulated vs. non-stimulated |
sCD40Lt stimulated |
| Effectors (PBMC) |
Overnight cultured vs 2 h cultured |
Overnight cultured cells |
| Effector:Tumor Ratio |
100:100, 100:200, 100:300K |
100:200K cells/well |
| 200:100, 200:200, 200:300K |
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| Incubation time of assay |
24 and 48 hours |
48 hour |
| Media |
5% and 10% HuAB vs. 5% and 10% FBS |
10% HuAB |
| Substrate Detection System |
AEC, TrueBlue™, and BCIP |
TrueBlue™ |
Malyguine et al. Journal of Translational Medicine 2004 2:9 doi:10.1186/1479-5876-2-9 |
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