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4. α4β1 (from HL60 lysate) was immobilised on a plate with a non-blocking anti-β1 antibody (TS2/16). The test compounds were titrated into a solution of 2-domain VCAM-Fc-Ig in a separate plate and added to the wells. The assay was carried out in TBS, 1% BSA, 1 mM MnCl2, 0.1% Tween. After incubation for 2 h at room temperature the plates were washed and residual VCAM visualised with peroxidase coupled anti-human Fc.
5. A Jurkat cell line expressing α4β1 was incubated at 37°C for 30 min with human 2-domain VCAM-1-FC immobilised on a plate with anti-human FC in the presence of the test compounds. The plates were washed and residual cells were stained with Rose Bengal.