Milliplex map magnetic mouse cytokine chemokine multiplex immunoassay

T cell antigen recall response was assessed in cell isolates from the spleen and cLN of immunized mice 2 wks after the final immunization (week 10). Methods for splenocyte and cLN lymphocyte preparation were previously described (38 (link)). For antigen recall, isolated cells were plated at a density of 8x10⁵cells/well and stimulated with 5 μg per well of recombinant S1 (Wuhan-Hu-1) in T cell media (DMEM, 5% FBS, 2 mM L-glutamine, 1% NEAA, 1 mM sodium pyruvate, 10 mM MOPS, 50 μM 2-mercaptoethanol, 100 IU penicillin, and 100 μg/mL streptomycin), in a total volume of 200 μL per well. Cells were stimulated for 72h at 37°C, and secreted cytokines (IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-17A, TNF-α, and IP-10) were measured relative to unstimulated cells in supernatants using a Milliplex MAP Magnetic Mouse Cytokine/Chemokine multiplex immunoassay (EMD Millipore).

T cell antigen recall response was assessed in cell isolates from the spleen and cLN of immunized mice 2 wks after the final immunization (week 10, or week 33 for longevity studies). Methods for splenocyte and cLN lymphocyte preparation were previously described^{15 (link), 16 (link)}. For antigen recall, isolated cells were plated at a density of 8x10⁵cells/well and stimulated with 5 μg/well RBD (WT) in T cell media (DMEM, 5% FBS, 2 mM L-glutamine, 1% NEAA, 1 mM sodium pyruvate, 10 mM MOPS, 50 μM 2-mercaptoethanol, 100 IU penicillin, and 100 μg/mL streptomycin), in a total volume of 200 μL for 72h at 37°C. Secreted cytokines (IFN-γ, IL-2, IP10, IL-4, IL-5, IL-6, IL-13, IL-10, IL-17A, and TNF-α) were measured relative to unstimulated cells in supernatants using a Milliplex MAP Magnetic Mouse Cytokine/Chemokine multiplex immunoassay (EMD Millipore).

T cell antigen recall response was assessed in cellular isolates obtained by processing the spleen and cLN of immunized mice 2 weeks after the final immunization (week 10, or week 33 for longevity studies). For antigen recall, isolated cells were plated at a density of 8 × 10⁵cells/well and stimulated with 5 μg/well RBD (WT) in T cell media (DMEM, 5% FBS, 2 mM L-glutamine, 1% NEAA, 1 mM sodium pyruvate, 10 mM MOPS, 50 μM 2-mercaptoethanol, 100 IU penicillin, and 100 μg/mL streptomycin), in a total volume of 200 μL for 72 h at 37 °C. Secreted cytokines (IFN-γ, IL-2, IP10, IL-4, IL-5, IL-6, IL-13, IL-10, IL-17A, and TNF-α) were measured relative to unstimulated cells in supernatants using a Milliplex MAP Magnetic Mouse Cytokine/Chemokine multiplex immunoassay (EMD Millipore).