Tumor-specific immune responses to IFN-γ were measured in vitro by ELISpot assay (Mouse IFN-γ ELISPOT Ready-SET-Go!; catalog no. 88-7384-88, eBioscience). A Millipore Multiscreen HTS-IP plate was coated overnight at 4°C with anti-mouse IFN-γ capture antibody. Single-cell suspensions of splenocytes were obtained from MC38 tumor-carrying mice and seeded onto the antibody-coated plate at a concentration of 2 × 105 cells per well. Cells were incubated with or without peptide sequence (KSPWFTTL) for 42 hours at 37°C and then discarded. The plate was then incubated with biotin-conjugated anti–IFN-γ detection antibody at room temperature for 2 hours, followed by incubation with avidin–horseradish peroxidase at room temperature for 2 hours. 3-Amino-9-ethylcarbazole substrate solution (catalog AEC101; Sigma-Aldrich, USA) was added for cytokine spot detection. Spots were imaged and quantified with a CTL ImmunoSpot Analyzer (Cellular Technology Ltd., USA).
Mouse ifn γ elispot ready set go
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Mouse IFN-γ ELISPOT Ready-SET-Go! is a laboratory assay kit designed for the quantitative determination of mouse interferon-gamma (IFN-γ) secreting cells. The kit contains the necessary components to perform an enzyme-linked immunospot (ELISPOT) assay, which is a widely used technique for the detection and enumeration of cytokine-secreting cells.
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5 protocols using mouse ifn γ elispot ready set go
1
IFN-γ ELISpot Assay for Tumor-Specific Immune Responses
2
Splenocyte IFNγ Secretion Assay
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Splenocytes from immunized mice were obtained as previously described and assayed for their ability to secrete IFNγ after in vitro stimulation with 5 µM of individual or pooled HIV-1 peptides using the ELISpot assay. The ELISpot assay was performed using mouse IFNγ ELISpot Ready-SET-Go! (eBiosciences) according to the manufacturer’s instructions. Spots were counted using an AID ELISpot Reader System (Autoimmun Diagnostika GmbH, Germany). The cutoff was 15 SFU per million splenocytes.
3
Evaluating ex-vivo DC priming via ELISPOT
4
IFN-γ ELISPOT Assay for Vaccine Response
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Splenocytes from immunized mice were assayed for their ability to secrete IFN-γ after in vitro stimulation with recombinant gp140 (5 μg/mL) using the ELISPOT assay. The ELISPOT assay was performed using mouse IFN-γ ELISPOT Ready-SET-Go! (eBiosciences) according to manufacturer’s instructions. Spots were counted using an automated stereomicroscope (KS ELISpot, Zeiss). The cutoff was 15 SFU per million splenocytes.
5
Enumerating IFNγ-secreting Cells in Mice
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Mice were immunized with live LVS (the immunization protocol is described in the next paragraph). Spleens of immunized mice were dissociated in GentleMACS C-tubes (Miltenyi Biotec, Bergisch Gladbach, Germany), filtered and separated on lympholyte-M media according to the manufacture protocol. Single-cell suspensions were seeded into 96-well ELISPOT plates (MAIPS4510, Merck Millipore, Ireland) in complete RPMI medium, supplemented with 10% heat-inactivated fetal calf serum, 1 mM Pen-Strep, nonessential amino acids, 2 mM l-glutamine, 1 mM sodium pyruvate, 25 mM HEPES, and 50 µM β-mercaptoethanol. Neutralized F. tularensis LVS bacteria were used as stimulating antigens at a concentration of 5 × 107 CFU equivalent per ml. Cells were plated for overnight incubation at a concentration of 106/well and serially diluted to enable single-spot enumeration. Each sample was tested in duplicate. IFNγ-secreting cells were enumerated (Mouse IFNγ ELISPOT Ready-SET-Go!, eBioscience, San Diego, CA, USA) according to the manufacturer’s protocol.
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