| Solution Information | help | |
| Enzyme: | Tumor necrosis factor | |
| inhibitor: | BDBM54363 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay materials: 1) HEK-293-T NF-kB-Luc cell line obtained from the assay provider's laboratory. 2) RNDsystems (Cat # 210-TA-010/CF) 3) SteadyGlo (Promega) (This assay was multiplexed with a cytotoxicity protocol described in AID 1849). Day 1 Procedure 1) Harvest HEK-293-T NFKB-Luc at 100% confluency 2) Add 1 uL/well NOD assay media with Multidrop 3) Spin down plates at 1000 rpm for 1 min in an Eppendorf 5810 centrifuge. 4) Serial compound dilutions: dispense 50nl 100% DMSO (columns 1-4, 47-48) or compounds (columns 5-46) using with Labcyte Echo 550 into plates from step 2. 5) Add TNFa to cell suspension at 0.1 ng/mL. 6) Seed 6000 cells/well in 4 uL/well to full plate HEK-293-T NFKB-Luc to Corning # 3727 white, 1536, hi-profile, TC-treated plate. 7) Spin down plates @ 500 RPM for 5 min on Eppendorf 5810 centrifuge. 8) Lid Plates. Sandwich 4 plates between 2 lidded 384 plates filled with H2O 9) Wrap plates securely in single layer of Plastic Wrap (Saran Wrap PVDC version). 10) | |
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