To detect and quantify the amount of IFN-γ secreting mouse splenocytes, harvested cells were used in an IFN-γ ELISpot assay. The assay was performed using the Mouse IFN-γ Development Module (R&D Systems, SEL485) according to the manufacturer's instructions. Mouse splenocytes were resuspended in complete RPMI medium and plated at a concentration of 2 × 105 cells/well. A set of peptides, each containing 15 aa residues overlapping by 11 aa spanning the entire protein consensus sequences of H1HA (A/Taiwan/1/86, A/Bayern/07/95, A/Texas/36/91, A/Beijing/262/95, A/New Caledonia/20/99, A/Solomon Islands/03/06, A/Brisbane/59/2007, A/Puerto Rico/8/34, A/South Carolina/1/18, A/California/07/09, and A/Mexico/InDRE4487/09) were synthesized from GeneScript (Piscataway, NJ). The set of peptides was pooled to make a concentration of 2 μg/ml per peptide and divided into 4 pools for use as stimulating antigens. As a positive control, cells were stimulated with 5 mg/ml Concanavalin A (Sigma-Aldrich, C5275). Complete RPMI medium was used as a negative control. Color development was performed using the ELISPOT Blue Color Module (R&D Systems, SEL002) according to the manufacturer's directions. An automated CTL Analyzer (Cleveland, OH) was used to count spots. The number of spot forming units (SFU) was reported as SFU/1 × 106 splenocytes.
Mouse ifn γ development module
Manufactured by R&D Systems
Sourced in United States
The Mouse IFN-γ Development Module is a kit that contains reagents for the development of an ELISA assay to quantify mouse interferon-gamma (IFN-γ) levels in biological samples.
Automatically generated - may contain errors
3 protocols using mouse ifn γ development module
1
IFN-γ ELISpot Assay for Mouse Splenocytes
2
Mouse IFN-γ ELISpot Assay
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The mouse IFN-γ ELISpot assay was performed by using Mouse IFN-γ Development Module (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions. Splenocytes were added in triplicate at an input cell number of 2 × 105 cells/well resuspended in RPMI medium 1640 with 10% fetal bovine serum. Splenocytes were stimulated with pools of 15mer peptides overlapping by 8 amino acids and spanning the length of each antigen. Peptides of NS3/4A, NS4B, and NS5A were synthesized by GenScript (Piscataway, NJ), resuspended in DMSO and pooled at a concentration of 1 mg/ml/peptide. Results were adjusted and graphed as the average number of spot forming units (SFU) per 1 × 106 splenocytes. Concavalin A (Sigma-Aldrich, St. Louis MO), at 4 mg/ml, was used as a positive control. The color development was performed according to the manufacturer’s instructions (ELISPOT Blue Color Module, R&D Systems, Minneapolis, MN). The spots on the plates were counted using an automated ELISPOT reader system.
3
Quantifying Tumor-Specific T-Cell Response
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Spleens from DC–tumor cell fusion vaccine‐treated mice were isolated 10 days after the last DC–tumor cell fusion vaccine injection. Splenocytes were isolated from the spleens as described previously. To stimulate splenocytes, B16‐F10 melanoma cells were treated with 15 μg·mL−1 mitomycin C (Nacalai Tesque Inc.) for 45 min. Splenocytes harvested from vaccine‐treated mice and mitomycin C‐treated B16‐F10 melanoma cells were mixed at a ratio of 10 : 1 and co‐cultured for 48 h. Nonadherent splenocytes were collected, and ELISpot assay was performed using the Mouse IFN‐γ Development Module (R&D Systems, Minneapolis, MN, USA) and the ELISpot Blue Color Module (R&D Systems). The numbers of IFN‐γ‐secreting cells were subsequently counted.
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