Single cell suspension was obtained from spleen, lymph nodes, bone marrow, synovium and resuspended in complete RPMI (ThermoFisher, #61870044) media containing 10% FCS (ThermoFisher, #26140079) and penicillin/streptomycin (Sigma, #P4333). Then, cells were added into COL2-coated (10 μg/ml), dsDNA-coated (20 μg/ml, Sigma-Aldrich, #D3664), nucleosome-coated (10 μg/ml, homemade) or anti-IgG-coated (1 μg/ml) ELISPOT plates (Merck Millipore, #MSIPS4W10). For dsDNA coating, Poly-l -Lysine (20 μg/ml, #P2658) was pre-coated one day before and dsDNA was coated in sterile TE buffer. After 2 h incubation at 37 °C, plates were washed and detected by biotinylated goat anti-mouse IgG (Southern Biotech, #1030-08), IgG1(Southern Biotech, #1070-08) or IgG2b (Southern Biotech, #1090-08), followed by ExtrAvidin® conjugated alkaline phosphatase (Sigma-Aldrich, #E2636) and BCIP/NBT (Sigma-Aldrich, #B5655). Spots were scanned with ImmunoScan and analyzed with ImmunoSpot software (Cellular Technology Ltd.).
Immunoscan
Manufactured by Cellular Technology
Sourced in United States
ImmunoScan is a laboratory instrument designed for the detection and analysis of various biomolecules, including proteins, antibodies, and other immunological markers. The core function of ImmunoScan is to perform automated immunoassays, providing precise and reliable data for research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Biotinylated goat anti mouse igg, by Southern Biotech (1 mentions)
Igg2b, by Southern Biotech (1 mentions)
Immunospot software, by Cellular Technology (1 mentions)
Bcip nbt, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Streptavidin ap, by R&D Systems (1 mentions)
Bcip nbt substrate, by R&D Systems (1 mentions)
Elispot plate, by Merck Group (1 mentions)
Antibody pair, by BD (1 mentions)
3 protocols using immunoscan
1
ELISPOT Assay for Antigen-Specific Antibodies
2
ELISpot Assay for Measuring Cytokine Secretion
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ELISpot plates (Millipore, Watford, UK) were coated overnight with IL-2 capture antibody (R&D Systems, Abingdon, UK), washed, and incubated overnight in blocking buffer (1% BSA in PBS). Cell density was adjusted to 4-6 x 10 6 PBMC/mL, and 100 μL of cells incubated with full length rHuPH20, A33 (positive control), or buffer for 8 days. ELISpot plates were washed, incubated with biotinylated anti-mouse detection antibody (R&D Systems, Abingdon, UK) for 1.5 h at 37°C, followed by incubation with streptavidin-AP (R&D Systems, Abingdon, UK) for 30 min at room temperature. Plates were then incubated with BCIP/NBT substrate (R&D Systems, Abingdon, UK) for 30 min at room temperature. The wells were washed with distilled H 2 0, dried, and scanned on an Immunoscan ® Analyser (Cellular Technology Limited, Cleveland, OH, USA) and spots per well were determined using Immunoscan ® software, Version 5.
3
IFN-gamma ELISPOT Assay Protocol
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A commercially available antibody pair (BD Biosciences) for the detection of IFN-g producing cells was used according to the manufacturer's protocol. ELISPOT 96 well plates were coated with anti-IFN-g antibody and subsequently blocked. Splenocytes were incubated with or without 10 mM of the respective peptide overnight at 37°C. After incubation with a biotinylated antibody for 2 hr, a streptavidin-alkaline phosphatase enzyme conjugate was added for 40 min at room temperature. Spots of the dried plate were counted using an ImmunoScan instrument (C.T.L., Cellular Technology Ltd.).
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