Concanavalin a (cona)

The culture medium used was RPMI-1640 (Gibco, Grand Island, NY, USA) supplemented with 10% FCS, 100 μg/ml streptomycin, and 100 IU/ml penicillin. Cell suspensions were made by pressing the thymuses and spleens through a cell strainer (Falcon, Franklin Lakes, NJ, USA). Cells were counted using a Coulter Counter (Coulter Electronics, Luton, UK). Suspensions of thymocytes and splenocytes were cultured at 10⁶ cells/ml culture medium with 5 μg/ml Concanavalin A (MP Biomedicals, Irvine, CA, USA) in 96-well tissue culture plates (Nunc, Roskilde, Denmark) for 24 hours. Culture conditions were 37°C in a humidified atmosphere containing 5% carbon dioxide.

The culture medium used was RPMI-1640 supplemented with 10% FCS, 100 μg/mL streptomycin, and 100 IU/mL penicillin. Cell suspensions were made by pressing the LNs through a cell strainer (Falcon, Franklin Lakes, NJ, USA). Cells were counted using a Coulter Counter. LN cell suspensions were cultured at 10
⁶cells/mL culture medium with 5 μg/mL Concanavalin A (MP Biomedicals, Irvine, CA, USA) in 96-well tissue culture plates (Nunc, Roskilde, Denmark) for 24 h. Spleen cell suspensions were cultured at 10
⁶cells/mL culture medium with 1 mg/mL OVA in 96-well tissue culture plates (Nunc) for 120 h. Culture conditions were 37 °C in a humidified atmosphere containing 5% CO
₂.

Spleens were removed from vaccinated animals one week after the second boost, and splenocytes isolated aseptically using standard methods. 1 X 10⁶ splenocytes per well were stimulated in 96 well round bottom plates for 72 hours with 20 μg endotoxin-free recombinant SML-4 or SML-5 protein. Concanavalin A (MP Biomedicals) was added at a concentration of 1μg/mL as a positive control stimulus, and media alone served as a negative control. Supernatants were analyzed by Luminex (ProcartaPlex kits; ThermoFisher) according to the manufacturer’s instructions. After 72 hours, splenocytes were transferred to a fresh 96 well flat-bottomed plate containing bound α-CD3 (clone 17A2, Biolegend) and cells were restimulated for 6 hours along with soluble α-CD28 (clone 37.51, Biolegend) with the addition of Brefeldin A (Biolegend) in the last 4 hours of stimulation. After blocking with addition of 10 μg/mL CD16/32 (clone 93, Biolegend), cells were then surface stained for CD4 (PerCP-Cy5.5, clone GK1.5, Biolegend), CD44 (Alexa Fluor 700, clone IM7, eBioscience) and zombie live/dead (BV 510, Biolegend). After fixation and permeabilization using Fix/Perm buffer (BD) cells were intracellularly stained for IFN-γ (PE, clone XMG1.2, Biolegend) and IL-4 (Pe-Cy7, clone BVD6-24G2, eBioscience). Cells were read on an LSRFortessa X-20 flow cytometer (BD) and data analyzed using FlowJo software.

Cytokine production by splenocytes was measured as previously described [25 (link)]. Briefly, immune cells (1.25 × 10⁶ cells/mL) were cultured for 48 h without mitogen (unstimulated cells) or with concanavalin A (ConA, 2.5 µg/mL; MP Biomedicals, Montreal, Quebec, Canada), lipopolysaccharide (LPS, 100 µg/mL, Sigma), or ovalbumin (OVA, 100 µg/mL, Sigma). Cells were then centrifuged for 10 min at 1000 rpm and the supernatants kept at −80 °C. Commercial ELISA kits were used to measure the concentrations of IL-1β, IL-2, IL-6, IL-10, TNF-α, TGF-β, and IFN-γ according to the manufacturer’s instructions and as described previously [25 (link)]. All detection limits were 15.63–4000 pg/mL except for IFN-γ which was 9.76–2500 pg/mL (R&D systems, Minneapolis, MN, USA). IL-13 and IL-5 was measured by sandwich ELISA commercial kits from MyBioSource (supplied by Cedarlane) as per detailed instructions. The detection range of both kits was 15.625 pg/mL–1000 pg/mL). Plasma concentrations of IgG and IgE ova-specific were measured according to the manufacturer’s instructions with a dilution 1:20 for both kits (Alpha Diagnostics Intl. supplied by Cedarlane). Absorbance was read on spectrophotometer and concentrations were calculated from the standard curve (SpectraMax 190, Molecular Devices Sunnyvale, CA, USA) with all measurements were conducted in duplicate with CV < 10%.