| Assay Method Information | |
| | Inhibition of α4β7 and α4β1 Integrins |
| Description: | MAdCAM (R&D Systems, 0.1 ug) was diluted in 50 μL PBS, added to each well of an opaque ELISA plate (Thermo), and incubated overnight at 4° C. Coated plates were washed once with PBS and then blocked with 200 μL of assay buffer (10 mM HEPES, 150 mM NaCl, 1 mM MnCl2, 0.1 mM CaCl2), 1% BSA) for 1 hour at 37° C. and 5% CO2. After incubation, buffer was aspirated from the plates and 50 μL of fresh assay buffer is added. Compounds were added by a compouns dispenser (Tecan) and DMSO concentration was normalized to 1% across each plate. 50 μL of RPMI-8866 cells (2×106 per mL) were added to each well and the plates were incubated for 1 hour at 37° C. and 5% CO2. After incubation, plates were allowed to cool to RT for 10 minutes before being washed four times in assay buffer by an automated plate washer (BioTek). After washing, 100 μL of a 1:1 mixture of assay buffer and CellTiter GLO 2.0 reagent (Promega) were added to each well. Plates were mixed for 2 minutes at 1000 rpm and then allowed to incubate an additional 10 minutes before reading out luminescence on a Tecan Spark plate reader. Raw data was converted to percent inhibition based on DMSO only and control compound wells, and curves were analyzed by 4-parameter fit within Dotmatics software. |
| Affinity data for this assay | |
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