Spleens were collected from mice intranasally vaccinated with PAQ11 or Q11 10 d after boost. Single-cell suspensions were prepared and plated at 0.5 × 106 cell per well (200 μL) in a 96-well plate (Millipore, Cat# MAIPSWU10) pre-coated with anti-mouse IFN-γ capture antibody (BD Bioscience, Cat# 51-2525KZ). The cells were then stimulated with soluble PA peptide (5 μM), or left untreated as negative controls, in a CO2 incubator at 37 °C for 48 h. To detect IFN-γ secreting cell spots, IFN-γ detection antibody (BD Bioscience, Cat# 51-1818KA), streptavidin-alkaline phosphatase (Mabtech, Cat# 3310-10), and substrate Sigmafast BCIP/NBT (Sigma, Cat# B5655) were applied sequentially following the manufacturer's protocol. Plates were imaged and IFN-γ spots were counted using an ELISPOT reader (Cellular Technology, Ltd).
Ifn γ detection antibody
Manufactured by BD
The IFN-γ detection antibody is a laboratory reagent used to detect the presence and measure the levels of the cytokine interferon-gamma (IFN-γ) in biological samples. It is a specific and sensitive tool for researchers studying immune response and inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin alkaline phosphatase, by Mabtech (2 mentions)
Anti mouse ifn γ capture antibody, by BD (2 mentions)
Elispot reader, by Cellular Technology (2 mentions)
Sigmafast bcip nbt, by Merck Group (2 mentions)
Ifn γ capture antibody, by BD (2 mentions)
Aec substrate, by BD (1 mentions)
Multiscreen ip filter plate, by Merck Group (1 mentions)
Elispot plate, by BD (1 mentions)
Human il 2, by Thermo Fisher Scientific (1 mentions)
Anti cd3ε, by BioLegend (1 mentions)
Anti cd28, by BioLegend (1 mentions)
Streptavidin hrp, by BD (1 mentions)
4 protocols using ifn γ detection antibody
1
IFN-γ ELISPOT Assay for Evaluating Murine Antigen-Specific T Cell Responses
2
Quantifying IFN-γ-Producing Splenocytes
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Spleens were aseptically removed 10 days after infection, and single-cell suspensions were made by the passage of the tissue through the sterile meshed after red blood cells were lysed. Multiscreen IP Filter Plates (Millipore Sigma, Burlington, MA, USA) were coated with 100 µl of 2 µg/ml of IFN-γ capture antibody (BD Biosciences) and incubated overnight at 4°C. After washing, wells were blocked with complete media for 2 hours at room temperature. Serial dilutions from 500,000 to 125,000 splenocytes were used with 2.5 µg/ml of protein and placed overnight at 37°C. The following day, wells were extensively washed and incubated with 100 µl of 1.25 µg/ml of IFN-γ detection antibody (BD Biosciences) overnight at 4°C. Finally, 1 µg/ml of streptavidin solution was added to the wells after washing and incubated for 1.5 hours at room temperature. The plates were developed by the addition of 100 µl of AEC substrate (1:50, BD Biosciences) for 30 minutes in the dark. Spots numbers were measure using an AID Classic ELISPOT Reader (AID-diagnostika GmbH, Strasberg, Germany).
3
IFNγ ELISpot Assay for Tumor-Specific T Cells
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ELISpot plate (BD Biosciences) was treated with sterile-filtered 70% ethanol before being washed three times with sterile PBS 1×. IFNγ capture antibody (BD Biosciences no. 551881) was plated and the plate was sealed and left to incubate at 4°C overnight. Positive control wells were also plated with anti-CD3ε (BioLegend no. 100340). The plate was washed three times with sterile PBS and blocked with 10% (v/v) FBS in PBS overnight at 4°C. IFNγ-stimulated 6694c2 or B16F10 cells were plated, except in the unstimulated and positive control wells. Pancreatic draining lymph nodes were excised from treated mice bearing orthotopic tumours, and lymph nodes were macerated to form a single-cell suspension. Lymph node cells (one-third of lymph node per well) were plated on top of pre-plated tumour cells with human IL-2 (PeproTech), and positive control wells also received anti-CD28 (BioLegend no. 102116). Plate was incubated at 37°C for 24 h before being washed with sterile water followed by PBST. IFNγ detection antibody (BD Biosciences no. 551881) was added, and plate was incubated for 2 h at room temperature. Wells were washed with PBST, and streptavidin-HRP (BD Biosciences) was added. Plate was incubated for 1 h at room temperature before being washed with PBST and PBS. AEC chromagen substrate (BD Biosciences) was added, and plate was developed. Plate was dried and analysed.
4
Quantifying PA-Specific IFN-γ Secreting Cells
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