| Solution Information | help | |
| Enzyme: | Tumor necrosis factor | |
| inhibitor: | BDBM67405 | |
| 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) TNF-a NF-kB induction assay: Dose Response Day 1 Procedure 1) Harvest HEK-293-T NF-kB-Luc at 100% confluency at 100% confluency 2) Supplement NOD plating media with DMSO to a final concentration of 0.62% DMSO 3) Dispense 3 uL (6000 cells)/well to every well of a 1536 TC-treated white plate (Corning # 3727). 4) Spin down plates at 500 rpm for 5 min in an Eppendorf 5810 centrifuge. 5) Serial compound dilutions: dispense with Labcyte Echo 550 50nl total volume 100% DMSO 6) Add 2 ul/well 0.25ng/mL dH20 TNF-a to wells in columns 3-48 using Multidrop. 7) Lid Plates. Sandwich 4 plates between 2 lidded 384 plates filled with H2O 8) Wrap plates securely in single layer of Plastic Wrap (Saran Wrap PVDC version). 9) Incubate overnight (14 hours) in 37 oC 5% CO2 incubator Day 2 Procedure 1) Add 3 ul/well SteadyGlo to each well wit | |
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