Anti cd3 pe cy5 antibody

Biotinylated ArtinM (20 μg/mL) was incubated for 45 min with D-mannose or D-galactose (1 mM, 10 mM, 20 mM, 30 mM, or 50 mM) or with medium alone. These mixtures were added to fixed (3 % formaldehyde-phosphate-buffered saline) spleen cells (1.5 × 10⁶/mL) that were then incubated for 30 min at room temperature. After two washes with phosphate-buffered saline, bound biotinylated ArtinM on spleen cells was reacted with streptavidin-fluorescein isothiocyanate (strp/FITC; 5 μg/mL; Invitrogen) for 40 min and fluorescence staining was analyzed with flow cytometry (Guava EasyCyte). The percentage of stained spleen cells was then determined. To evaluate ArtinM binding on T cells, we treated the spleen cells with bound ArtinM for an additional 40 min with anti-CD3 PE-Cy5 antibody (4 μg/mL; BD Biosciences) and performed flow cytometry analysis. The percentage of double-positive cells for each experimental condition was then analyzed.

The binding of ArtinM to CD3 receptor was determined on CD4⁺ T cells isolated from spleen cell suspensions using CD4⁺ T cell isolation kits II and MS columns from Miltenyi-Biotec according to the manufacturer’s instructions. In the first assay, the cells were fixed and incubated with ArtinM (25 μg/mL) for 40 min at 4 °C. After two washes with phosphate-buffered saline (PBS), the cells were incubated with an anti-CD3 PE-Cy5 antibody (10 μg/mL; clone 17A2; BD Biosciences) for 40 min at 4 °C. The percentage of labeled cells was determined using flow cytometry (Guava EasyCyte). A functional inhibition assay was carried out by pre-incubating CD4⁺ T cells with either the anti-CD3 antibody (5 μg/mL) or an anti-CD28 antibody (5 μg/mL; clone 37.51; BD Biosciences) for 40 min at 4 °C. After washing with RPMI medium, the cells were stimulated for 48 h with jArtinM (78 nM). The culture supernatants were tested by means of ELISA for IL-2.