Feline and Murine IFNγ ELISPOT Assay

Mouse splenocytes or feline PBMCs isolated by ficoll separation (GE Healthcare) were maintained in Roswell Park Memorial Institute medium supplemented with fetal bovine sera, penicillin/streptomycin. Feline PBMCs were supplemented with 0.5 ng/mL feline IL2 (R&D Systems). Cells were plated at 2x10⁵/well in round bottom 96-well plates and cultured with 10 μg/mL feHER2-Fc (3T3 supernatant-equivalent as described above), huHER2-Fc, human IgG control (Jackson Immunolabs) or control 3T3-conditioned medium for 48 (mouse) or 72 (feline) hours. Total well contents were then transferred to mouse or feline (R&D Systems) IFNγ ELISPOT plates and incubated for an additional 48 hours prior to detection and enumeration as previously described (44 (link)) or per manufacturer protocol. Visualized cytokine spots were enumerated using the ImmunoSpot analyzer (CTL, Shaker Heights, OH) and expressed as the number of cytokine-producing cells per 10⁶ splenocytes or PBMCs.

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Gibson H., Veenstra J., Jones R., Vaishampayan U., Sauerbrey M., Bepler G., Lum L., Reyes J., Weise A, & Wei W.Z. (2015). Induction of HER2 Immunity in Outbred Domestic Cats by DNA Electrovaccination. Cancer immunology research, 3(7), 777-786.

Publication 2015

ficoll separation IFNγ ELISPOT plates

Cells Conditioned medium Cytokine Elispot Feline Fetal bovine sera Ficoll Human Ifnγ Mouse Penicillin Spots Streptomycin

Corresponding Organization :

Other organizations : The Barbara Ann Karmanos Cancer Institute, Wayne State University, Michigan Reproductive Medicine

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Variable analysis

independent variables

FeHER2-Fc (3T3 supernatant-equivalent)
HuHER2-Fc
Human IgG control (Jackson Immunolabs)
Control 3T3-conditioned medium

dependent variables

Number of cytokine-producing cells per 10^6 splenocytes or PBMCs

control variables

Mouse splenocytes or feline PBMCs isolated by ficoll separation (GE Healthcare)
Roswell Park Memorial Institute medium supplemented with fetal bovine sera, penicillin/streptomycin
Feline PBMCs supplemented with 0.5 ng/mL feline IL2 (R&D Systems)
Cells plated at 2x10^5/well in round bottom 96-well plates
Cells cultured for 48 (mouse) or 72 (feline) hours

positive controls

Human IgG control (Jackson Immunolabs)

negative controls

Control 3T3-conditioned medium

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