Facscalibur cell analyzer

FITC-labeled Gal-1 (1 μM) or FITC-labeled Gal-1 that was preincubated with scrambled or AP-74 M-545 aptamer (5 and 10 μM) was added to Jurkat T cells for 60 min at 4°C. These FITC signals of Gal-1-treated Jurkat T cells were further washed and analyzed by a FACSCalibur cell analyzer (BD). In the T cell apoptosis assay, Gal-1 (1.3 μM), which was preincubated with scrambled or AP-74 M-545 (2.5, 5, and 10 μM) aptamer, was added to 1 × 10⁵ Jurkat T cells for 12 h at 37°C. The Gal-1-treated Jurkat T cells were collected and stained by an FITC annexin V/PI kit (Invitrogen). The staining T cells were washed and analyzed by a FACSCalibur cell analyzer (BD).

Immunofluorescent staining was performed for DC-surface phenotypic analysis as described previously [6 (link)]. DCs were stained in fluorescence-activated cell sorting (FACS) buffer (FACS flow, BD Bioscience, San Jose, CA, USA) with the appropriate antibodies and incubated at 4℃ for 20 minutes. FITC-labeled rat anti-mouse CD14 (rmC5-3), anti-mouse CD86 (GL1), anti-mouse I-A/I-E (2G9), anti-mouse H2-d (M1/42), PE-labeled hamster anti-mouse CD11c (HL3), anti-mouse CD80 (16-10A1), rat anti-mouse CD40 (3/23) with PE-or FITC-labeled isotype control antibodies were purchased from BD Pharmingen and BioLegend. After washing with FACS staining buffer, cells were analyzed using the BD FACSCalibur cell analyzer. For intracellular staining of SHB, cells were pre-stained with FITC-labeled CD11c antibody, then fixed and permeabilized using the BD Cytofix/Cytoperm kit (BD Bioscience Pharmingen). Cells were then incubated with SHB rabbit polyclonal antibody (Santa Cruz Biotechnology) for 1 hour, and stained with FITC-labeled secondary goat anti-rabbit IgG antibody. The stained cells were washed with BD Perm/wash buffer and analyzed with flow cytometry.