Antigen-specific IFNγ-producing cells in the spleen were determined by the ELISPOT. Lymphocytes were separated from the spleen as described above and seeded at a concentration of 0.8 × 106 per well on mouse IFNγ pre-coated PVDF plates (DAKEWE Biotech). Cells were then stimulated with 100 µg/mL OVA or 40 µg/mL ESAT-6 at 37°C for 16 h. Spot forming units were determined with ELISPOT plate reader (Cellular Technology).
Elispot plate reader
Manufactured by Cellular Technology
Sourced in United States
The ELISPOT plate reader is a laboratory instrument designed to automatically detect and quantify cytokine-secreting cells in cell samples. It utilizes an ELISPOT assay, which is a sensitive technique for the detection and enumeration of individual cells that secrete specific proteins, such as cytokines. The plate reader precisely measures and records the number of spots, which correspond to the number of individual cells secreting the protein of interest.
Automatically generated - may contain errors
Lab products found in correlation
Mouse ifn γ elispot kit, by BD (3 mentions)
Mouse elispot kit, by BD (2 mentions)
Phytohemagglutinin (pha), by Merck Group (2 mentions)
Ifn γ elispot assay, by Dakewe (1 mentions)
Dual color ifn γ il 4 elispot kit, by R&D Systems (1 mentions)
Multiscreen hts filter plates, by Merck Group (1 mentions)
Anti ifn γ antibody clone r4 6a2, by BD (1 mentions)
Ifn γ elispot assay, by Mabtech (1 mentions)
Ova257 264 peptide, by AnaSpec (1 mentions)
10 protocols using elispot plate reader
1
Quantify Antigen-Specific IFNγ Cells
2
Evaluating IFN-γ-producing Cells in Spleen
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The frequencies of IFN-γ-producing cells in the spleen were evaluated using mouse IFN-γ ELISPOT kits (BD). Spots were counted using an ELISPOT plate reader (Cellular Technology Ltd) as described previously11 (link)23 (link).
3
IFN-γ ELISPOT Assay for HTNV Peptide Immunogenicity
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The IFN-γ ELISPOT assay (Dakewe Biotech Company Ltd, Shenzhen, China) was used for the determination of the immunogenicity of the HTNV linear multi-epitope peptide ex vivo [21 (link)]. Briefly, the splenocytes isolated from immunized HLA-A2.1/Kb Tg mice in each group were placed on ELISPOT plates (IFN-γ mAb precoated), at 1 × 106 cells/well. Cells were stimulated with the single HTNV CTL epitope (10 μM) for 24 h at 37 °C. Phytohemagglutinin (PHA, 10 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) stimulation served as a positive control, while the irrelevant peptide and media alone served as negative controls. Spots developed after incubation for 10–30 min with 3-amino-9-ethylcarbazole (AEC) substrate in the dark and counted with an ELISPOT plate reader (Cellular Technology Limited, USA). The number of spots was expressed as adjusted spot-forming cells (SFC)/106 splenocytes after subtracting the average negative values. The positive response was defined as at least 50 SFC/106 input cells, exceeding 3-times the background response. The SFC/106 splenocytes in the unstimulated control wells never exceeded 5 spots per well.
4
ELISpot Monitoring of Cross-Protective Responses
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Dual-Color IFN-γ/IL-4 ELISpot Kit (R&D Systems# ELD5217) was adopted for monitoring cross-protective responses of PMSB-inoculated hosts to CSC subsets. Briefly, thymocytes were harvested from host thymus 2 weeks after the boost. 5 × 104 recipient thymocytes as responder cells and 3 × 103 PMSB cells, or tumor spheroid cells as stimulators were respectively performed according to the manufacturer's protocol with common PSCs as stimulator control. Spots were automatically scanned and enumerated using ELISpot plate reader (Cellular Technology Ltd., Cleveland, OH).
5
Evaluating Antigen-Specific IFN-γ Responses
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The frequency of IFN-γ-producing cells was evaluated using mouse IFN-γ ELISPOT kits (BD Bioscience). Briefly, the splenocytes were plated at 5 × 105 cells/well onto purified IFN-γ Ab-coated ELISPOT plates and stimulated with 1 μg/mL OVA257–264 or 500 TCID50/mL of UV-inactivated pH1N1 virus for 60 h at 37 °C. The SFUs were enumerated using an ELISPOT plate reader (Cellular Technology Ltd., OH, USA).
6
Quantifying Mtb-specific IFNγ Cells in Lungs
7
Evaluating Antigen-Specific IFN-γ Responses using ELISPOT
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Antigen-specific IFN-γ-producing spot forming units (SFUs) were evaluated using mouse IFN-γ ELISPOT kits (BD Biosciences). Splenocytes were plated at 1 × 106 cells/well onto purified IFN-γ antibody-coated PVDF-backed plates and stimulated with an RBD peptide pool (2 μg/mL each), 5 μg/mL individual peptide, or 5 μg/mL OVA257–264 at 37 °C for 2 days. In a separate experiment, splenocytes were stimulated with 10-mer peptides (5 μg/mL) in the presence of mouse IgG2a isotype as a control, anti-H2Kb and/or anti-H2Db mAbs (20 μg/mL) at 37 °C for 2 days. The IFN-γ-positive SFUs were enumerated using an ELISPOT plate reader (Cellular Technology Ltd., Cleveland, OH, USA).
8
Immunogenicity Assessment of HTNV-NP 9-mer Peptide
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The determination of the immunogenicity of the HTNV-NP 9-mer peptide in vivo was performed using an interferon (IFN)-γ ELISPOT assay (Mabtech, Büro Deutschland, Germany), as previously described (20 (link)). Briefly, the splenocytes obtained from HLA-A2.1/Kb Tg mice were placed in duplicate on ELISPOT plates at 1 × 106 cells/well and stimulated overnight with peptides (10 μM). Cells with phytohemagglutinin (PHA, 10 μg/ml, Sigma-Aldrich, St. Louis, MO, USA) or no peptide stimulation served as positive and background controls, respectively. Spots formed by the deposition of the enzyme substrate were counted using an ELISPOT plate reader (Cellular Technology Limited, USA). Adjusted spot-forming cells (SFC) after subtracting the average negative values are expressed as SFC/106 splenocytes. The positive response was defined as at least 50 SFC/106 input cells, exceeding three times the background response after subtracting the number of spots in the background controls from those in the stimulated samples. The SFC/106 splenocytes in the unstimulated control wells never exceeded five spots per well.
9
Measurement of Antigen-Specific IFN-γ Response
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The frequencies of antigen-specific IFN-γ-producing cells were evaluated using a mouse ELISPOT kit (BD Bioscience), as previously described (18 (link), 19 (link)). Briefly, splenocytes were obtained from immunized mice and plated at 5 × 105 cells/well onto purified anti-IFN-γ-coated ELISPOT plates. The cells were stimulated with OVA257−264 peptide (0.5 μg/well; Anaspec) or UV-inactivated viruses, including 500 TCID50/well of A/California/04/09 (pH1N1), 2,000 TCID50/well of A/Puerto Rico/8/34 (H1N1), or 2,000 TCID50/well of H3N2 viruses for 3 days. The spot-forming units (SFUs) of IFN-γ-producing cells were counted using an ELISPOT plate reader (Cellular Technology Ltd).
10
Quantifying Antigen-Specific IFN-γ Response
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The frequencies of antigen-specific IFN-γ-secreting cells were evaluated using a mouse ELISPOT kit (BD Biosciences), as previously described [12 (link),23 (link)]. Briefly, 14 days after the last vaccination, splenocytes were obtained from the immunized mice and then plated at 5 × 105 cells/well onto purified anti-IFN-γ-coated ELISPOT plates. The cells were treated with 0.5 µg/well of OVA257–264 peptides (Anaspec, San Jose, CA, USA) or the 500 median tissue culture infectious dose (TCID50)/well of UV-inactivated pH1N1 influenza virus for 3 days at 37 °C. The spot-forming units (SFUs) of antigen-specific IFN-γ-secreting cells were calculated using an ELISPOT plate reader (Cellular Technology Ltd., Cleveland, OH, USA).
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