ICS and ELISpot were performed using splenocytes as described previously (25 (link)). For ICS, the splenocytes were stimulated for 6 h with a final concentration of 1 μg/mL of the immunodominant CD8+ T-cell epitope NYDNAGTNL (PfCSP39−47) and 1 μg/mL of GolgiPlug™ (BD Biosciences, Tokyo, Japan) in a 96-well U-bottom tissue culture plate (Corning Inc.). The cells were then surface stained with anti-mouse CD16/32 antibody, Pacific Blue™-conjugated anti-mouse CD4 antibody, and PerCP/Cy5.5-conjugated anti-mouse CD8β antibody, and the cytokine was stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IFN-γ antibody or a FITC-conjugated rat IgG1κ isotype control antibody. Data were acquired with a BD FACSVerse™ Flow Cytometer (BD Biosciences) and analyzed with FlowJo (Tree Star, Ashland, OR, USA). All antibodies used in these experiments were purchased from BioLegend (San Diego, CA, USA).
For ELISpot assays, splenocytes were cultured for 20–24 h on an ELISpot microplate (PerkinElmer, Yokohama, Japan) with the H-2Kd-restricted PfCSP T-cell epitope (NYDNAGTNL, PfCSP39−47; final concentration, 1 μg/mL) or the PfCSP-overlapping peptide pool (final concentration, 5 μg/mL). Results are expressed as IFN-γ spot-forming units (SFU) per million splenocytes.
For ELISpot assays, splenocytes were cultured for 20–24 h on an ELISpot microplate (PerkinElmer, Yokohama, Japan) with the H-2Kd-restricted PfCSP T-cell epitope (NYDNAGTNL, PfCSP39−47; final concentration, 1 μg/mL) or the PfCSP-overlapping peptide pool (final concentration, 5 μg/mL). Results are expressed as IFN-γ spot-forming units (SFU) per million splenocytes.
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