Concanavalin a from canavalia ensiformis

Agglutinating activity of AsCTL-42 was assessed as described previously [18 (link),20 (link)], using S. Typhimurium strain 4/74. Bacteria grown in Luria–Bertani (LB) medium were collected at mid-logarithmic phase by centrifugation at 880× g for 5 min. They were then washed and re-suspended in tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) at approximately 10⁹ cells/mL. Twenty microliters of bacterial suspension were mixed with 20 µL treatments (diluted in TBS) with or without added calcium (10 mM CaCl₂) and incubated for 1 h at room temperature on a glass slide. Concanavalin A from Canavalia ensiformis (Sigma-Aldrich, St. Louis, MO, USA) was included as a positive control. Samples were visualized and photographed using the 40× objective (final 400× magnification) on a Leica DM750 microscope equipped with an ICC50HD digital camera (Leica Microsystems, Wetzlar, Germany).

Viable spleen cells were thawed and suspended in DPBS at a concentration of 5 × 10⁶ cells/mL and labeled with 2.5 µM carboxyfluorescein succinimidyl ester (CFSE) by applying the CellTrace™ CFSE Cell Proliferation kit (Invitrogen), according to previous reports [36 (link)]. After CFSE labeling, splenocytes were resuspended in complete RPMI 1640 medium, plated in 24-well plates (5 × 10⁶ cells/well). Subsequently, the cells were stimulated in vitro by adding 8000 TCID₅₀/mL of the three vaccine viruses (H1N1, H1N2 and H3N2) for 96 h at 37 °C, under 5% CO₂, in the dark. For the negative control, only culture medium was added to cells (non-virus-stimulated cells), and for the positive control, the cells were stimulated separately with 5 µg/mL of Concanavalin A from Canavalia ensiformis (Sigma-Aldrich). After in vitro stimulation of cells with H1N1, H1N2 and H3N2 viruses, lymphocyte proliferation from spleens was measured as an indicator of T and B-cell responses at 21 days after the boost immunization.