| Solution Information | help | |
| Enzyme: | Integrin alpha-L | |
| inhibitor: | BDBM79604 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | The assay was conducted in a 96-well microplate format. Drosophila micro-vesicles expressing B7-1 (the ligand partner for T cell-expressed CD28), ICAM-1 (the ligand partner for T cell expressed LFA-1) and the Ld class I murine MHC molecule, are incubated with the antigenic peptide, QL9 (QLSPFPFDL, single letter amino acid sequence), that binds to Ld and is specifically recognized by 2C TCR transgenic CD8+ T cells. QL9-loaded micro-vesicles are incubated (1.5 hour/37degrees C) with MACS-purified CD8+ T cells, followed by anti-CD8 (Alexa647) and anti-B7-1 (PE) staining (20 minutes on ice). The stained, CD8+ T cell/micro-vesicle mixture is washed twice by centrifugation to remove excess fluorescent antibody, resuspended in culture media and analyzed by flow cytometry using the HyperCyt (Intellicyt, USA) high-throughput analytical platform. Negative control wells contain anti-LFA-1 (5microG/milliL; clone M17/4) that blocks the binding of QL9-loaded micro-vesicles to CD8+ T cells. Positiv | |
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