Complete freund s adjuvant cfa

The production of anti-mullet immunoglobulin in mice was performed according to Sharon et al. [31 (link)]. In short, 200 µL blood was withdrawn from the caudal vein of three anesthetized sick mullets (presenting higher levels of antibodies) using 1 mL syringes with a 30× g needle. Blood samples were incubated overnight at 4 °C and then centrifuged at 3500× g at 4 °C for 30 min; 100 µL of mullet serum was transferred to a new tube and the rest was frozen at −80 °C. The 100 µL of mullet serum was incubated with 40 µL of protein A/G covalently immobilized on Sepharose beads (ABCam, Cambridge, UK) and shaken for 2 h at room temperature (RT). The protein A/G beads were washed three times in PBS and centrifuged at 14,000× g for 1 min at RT; the supernatant was discarded, and the pellet with the proteins was resuspended in PBS at a final volume of 200 µL and emulsified (1:1, v/v ratio) in complete Freund’s adjuvant (CFA, Invivo Gen, Toulouse, France).

C57BL6 mice were immunized against OVA (50 μg – Grade IV, Sigma Aldrich) in Complete Freund's Adjuvant (CFA – InvivoGen). After 7 days, MutuDCs were pulsed with 100 μg/mL of OVA overnight, washed with PBS twice, and 3 × 10⁶ cells were transferred to immunized mice by intraperitoneal (i.p.) injection. When IL10-DCs and IL35-DCs were transferred in combination, they were mixed only a few minutes before the injection, at 1:1 ratio. One week later, mice were challenged with 25 μL of heat-aggregated OVA (20 mg/mL – 500 μg/animal) in one footpad and the same volume of PBS was injected in the contralateral footpad as a control. Footpad thickness was measured with a dial thickness gauge (Mitutoyo) multiple times for 72 h. Blood, lymph nodes and spleen were collected and processed as mentioned above. Total cells were re-stimulated ex vivo with 100 μg/mL OVA for 24 h.

Isolated murine and human cells were cultured with sterile RPMI 1640 supplemented with L-Glutamine, Penicillin, Streptomycin, 25mM Hepes (Lonza, Walkersville, MD).
Nickel (II) chloride (NiCl₂), cobalt (II) chloride (CoCl₂), phorbol 12-myristate 13-acetate (PMA), ionomycin, phytohemagglutinin (PHA), and Mitomycin-C were purchased from Sigma Aldrich (St. Louis, MO). NiCl₂ and CoCl₂ were reconstituted in sterile water and stock solutions were freshly prepared for each in vitro and in vivo experiment. Complete freund’s adjuvant (CFA) was purchased from InvivoGen (San Diego, CA).