Crpmi

Blood samples from the retro-orbital sinus were heparinized to prevent clotting. Blood volumes were recorded and then blood was centrifuged to collect and archive plasma for quantification of antibodies. Spleens and lymph nodes (inguinal and brachial) were harvested in Hanks’ Balanced Salt Solution (HBSS; GE Healthcare Life Sciences). Samples were kept on ice during transportation and processing. Single-cell suspensions were made by pressing spleens and lymph nodes between the frosted ends of glass slides. Erythrocytes in blood and spleens were lysed. Cells were counted using an improved Neubauer counting chamber using trypan blue dye exculsion to assess viability, which was consistently >90%. All cells were plated in RPMI-1640 medium containing 10% heat-inactivated bovine calf serum and penicillin/streptomycin (cRPMI; GE Healthcare Life Sciences) in 96-well round-bottom plates and were maintained at 4 °C during handling and centrifugation (500 g). Up to two million cells were added to each well. Samples were processed by the same individual to reduce technical variability and they were blinded to treatment groups to eliminate bias.

Blood sampled from the retro-orbital sinus was heparinized to prevent clotting, and the volumes were recorded. Samples were kept on ice during transportation and processing. Single-cell suspensions were made by pressing spleens between the frosted ends of glass microscope slides. Erythrocytes in blood and spleens were lysed. Cells were counted using an improved Neubauer counting chamber using trypan blue dye exclusion to assess viability, which was consistently >90%. All cells were plated in Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% heat-inactivated bovine calf serum, 0.1% 2-mercaptoethanol, and penicillin/streptomycin (cRPMI; GE Healthcare Life Sciences) in 96-well round-bottom plates and were maintained at 4 °C during handling and centrifugation (500× g). Up to two million cells were added to each well of a 96-well plate. Samples were processed by the same individual to reduce technical variability.

Splenocytes were seeded (2 × 10⁵/well) and stimulated with the NG-34 epitope (5 µg/mL); media cRPMI (as negative control) and concavalin A (1 µg/mL) (as positive control) (GE Healthcare, Marlborough, MA, USA). The supernatants were harvested after 72 h incubation at 37 °C and a 9-plex panel MSD standard Th1/Th2/Th17 which checks IFN-γ, TNF-α, IL- 5, IL-10, IL-13 and IL-17 cytokines together with two U-plex panels for IL-6 and IL-1β detection were ran following the manufacturer’s instructions (MSD, Rockville, MD, USA).