CD4+ T cells were isolated from spleen of C57Bl/6 or Itgb3−/− mice by negative selection and magnetic sorting (Miltenyi Biotech). To generate Th17 cells, T cells were cultured in RPMI 1640 medium supplemented with 10% FCS, IL-6, IL-23, IL-1β, and anti-IFN-γ antibody, with or without TGF-β1, in plates coated with anti-CD3 antibody. Cells were harvested between days 5–7 of culture and resuspended in migration medium (RPMI with 2% BSA). Migration was assayed in 96-well cell-permeable chambers (5-μm pore size polycarbonate membranes) which had been pre-coated by incubation with rat fibronectin (Sigma; 10 μg/ml), vitronectin (Sigma; 1 μg/ml) or no matrix at 37°C for 2 hr, and remaining protein binding sites blocked with RPMI/BSA for 1 hr. 4 × 104 T cells were added to the upper chambers, and RPMI containing 1% FCS was added to the lower chambers. In some cases, GRGDNP peptide (Enzo Life Sciences) was added to upper chambers at 2 μg/ml. After 2 hr, the upper chambers were removed, and migrated cells in the lower chamber were stained with Calcein AM (1 μM; Invitrogen) and visualized and counted by imaging plate reader (Cytation 3; BioTek).
Grgdnp peptide
Manufactured by Enzo Life Sciences
The GRGDNP peptide is a synthetic peptide that contains the amino acid sequence Glycine-Arginine-Glycine-Aspartic acid-Asparagine-Proline. It is a commonly used tool in cell biology and biochemistry research.
Automatically generated - may contain errors
2 protocols using grgdnp peptide
1
Th17 Cell Migration Assay
2
Th17 Cell Migration Assay
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CD4+ T cells were isolated from spleen of C57Bl/6 or Itgb3−/− mice by negative selection and magnetic sorting (Miltenyi Biotech). To generate Th17 cells, T cells were cultured in RPMI 1640 medium supplemented with 10% FCS, IL-6, IL-23, IL-1β, and anti-IFN-γ antibody, with or without TGF-β1, in plates coated with anti-CD3 antibody. Cells were harvested between days 5–7 of culture and resuspended in migration medium (RPMI with 2% BSA). Migration was assayed in 96-well cell-permeable chambers (5-μm pore size polycarbonate membranes) which had been pre-coated by incubation with rat fibronectin (Sigma; 10 μg/ml), vitronectin (Sigma; 1 μg/ml) or no matrix at 37°C for 2 hr, and remaining protein binding sites blocked with RPMI/BSA for 1 hr. 4 × 104 T cells were added to the upper chambers, and RPMI containing 1% FCS was added to the lower chambers. In some cases, GRGDNP peptide (Enzo Life Sciences) was added to upper chambers at 2 μg/ml. After 2 hr, the upper chambers were removed, and migrated cells in the lower chamber were stained with Calcein AM (1 μM; Invitrogen) and visualized and counted by imaging plate reader (Cytation 3; BioTek).
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