B and T cell-mediated immunity to HspA and MV antigens was analyzed on isolated spleen lymphocytes of immunized Ifnarko-CD46Ge mice using mouse IFN-γ and IgM/IgG dual-color B cell ELISPOT kits (R&D Systems). Spleen cells were harvested, washed, and plated (5 × 106 cells/well in 1 mL of culture medium) in 24-well plates for stimulation with MV-NIS at an MOI of 0.1, 100 ng/mL purified HspA, and corresponding empty pET-28a control extracts overnight. On the next day 5 × 104 and 5 × 105 cells in 100 μL of medium were transferred to the IFN-γ ELISPOT plates and incubated for 24 or 72 h before the analysis. For the HspA-specific IgM/IgG B cell response detection, spleen cells were transferred to HspA (200 ng/well) or control antigen-coated ELISPOT plates and cultured for 3 days. The reaction was developed following the ELISPOT kit instructions (R&D Systems). Results were analyzed on an ImmunoSpot S4 Pro analyzer (Cellular Technology, Cleveland, OH, USA) using ImmunoSpot version 4.0 software (Cellular Technology).
Immunospot version 4
Manufactured by Cellular Technology
Sourced in United States
The ImmunoSpot version 4.0 software is a digital analysis tool designed for use with the ImmunoSpot instrumentation. The software provides automated counting and analysis of immune cell spots generated from various assays.
Automatically generated - may contain errors
2 protocols using immunospot version 4
1
Analyzing B and T Cell Immunity to HspA and MV
2
Quantifying IFNγ-Producing Cells by ELISPOT
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Human IFNγ ELISPOT kits (R&D Systems; Minneapolis, MN) were used to measure the number of IFNγ-producing cells, as previously described [16 (link),44 (link),45 (link)], following the manufacturer’s protocol. We stimulated subjects’ PBMCs (or, alternatively, left them unstimulated) in triplicate with the Edmonston strain of MV (multiplicity of infection, MOI = 0.5), and developed the reaction after 42 h incubation at 37 °C, in 5% CO2. PHA (5 μg/mL) was used as a positive control. All plates were scanned and analyzed using the same counting parameters on an ImmunoSpot® S4 Pro Analyzer (Cellular Technology Ltd.; Cleveland, OH, USA) using ImmunoSpot® version 4.0 software (Cellular Technology Ltd.). The ELISPOT response is presented in spot-forming units (SFUs) per 2 × 105 cells (median MV-specific stimulated response from triplicate measurements, minus median unstimulated response from triplicate measurements).
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