ELISpot assays were performed as previously reported (26 (link)). Briefly, 96-well ELISpot plates were coated with a purified anti-mouse IFN-γ capture antibody (BD ELISPOT Set, USA) and incubated overnight at 4°C. The following day, 6×106 splenocyte suspensions from each group were stimulated with 8 μg/μL of either C (ACLVGDKVM), E1 (HSMTNAVTI), E2 (IILYYYELY) dominant single peptides (27 (link), 28 (link)), or Con A (positive control), in the presence of a positive control. The plates were then incubated at 37°C in 5% CO2 for 20–24 h. The next steps were performed according to the manufacturer’s instructions (BD ELISPOT Set, USA). A spot forming unit (SFU) was used to represent a T cell-secreting IFN-γ. The plates were then detected using an ELISpot plate reader (Biosys, So. Pasadena, CA).
Elispot set
Manufactured by BD
Sourced in United States
The BD ELISPOT Set is a laboratory equipment used for the detection and enumeration of antigen-specific T cells or B cells that secrete cytokines or antibodies. It provides a platform for the analysis of cellular immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Elispot kit, by R&D Systems (1 mentions)
Aec substrate set, by BD (1 mentions)
Immunospot reader, by Cellular Technology (1 mentions)
Akp streptavidin, by BD (1 mentions)
Bcip nbt liquid substrate system, by Merck Group (1 mentions)
Elisa kits for il 6, by BD (1 mentions)
Il 23, by Thermo Fisher Scientific (1 mentions)
9 protocols using elispot set
1
IFN-γ ELISpot Assay for T Cell Response
2
Quantifying Mucosal Immune Responses
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Mixed cellulose ester membrane-bottom plates (Millipore) were coated with rT2544 or anti-mouse total immunoglobulin overnight at 4 °C. Wells were blocked with 1% BSA and incubated for 2 h at 37 °C. Cells isolated from mesenteric lymph nodes (MLN), Peyer’s Patches (PP), and spleen of immunized mice was added to blocked wells for 5 h, followed by washing with PBST. The plate was incubated with enzyme-conjugated anti-mouse IgG and IgA (Southern Biotech) overnight at 4 °C and developed with substrate. The number of spots was counted separately for each well. In a separate experiment, IFN-γ (ELISpot set from BD Bioscience) and IL-17 (ELISpot kit from R&D) pre coated mixed cellulose ester membrane-bottom plates were incubated with cells isolated from Peyer’s Patches (PP) stimulated with rT2544 for 24 h. The plate was incubated with enzyme-conjugated anti-mouse IFN-γ and IL-17 antibodies and developed with substrate. The number of spots was counted separately for each well.
3
Murine IFN-gamma ELISpot Assay
4
Measuring T Cell IFN-γ Response to SARS-CoV-2 Peptides
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The peptide pools spanning the entire S/N/E/M protein as consecutive 15-mers overlapping by 10 amino acids were synthesized by Scilight Biotechnology, LLC. Approximately 2.5 mg of each purified peptide in the peptide pool was present per vial. The experiment was conducted as described previously [18 (link)]. Briefly, 96-well plates (BD ELISPOT Set, USA) were coated with anti–IFN-γ capture Ab and incubated overnight at 4 °C. The plates were blocked with the complete culture medium after washing three times. Splenocytes were harvested after the mice were euthanized on days 35 and 120 Fresh single-cell suspensions from each group were plated at 5 × 106 per well, and peptides were added. The plates were then incubated at 37 °C in 5% CO2 for 22 h and detected using an ELISpot plate reader (Biosys, So. Pasadena, CA, USA). A spot-forming unit (SFU) represents a T cell-secreting IFN-γ.
5
Quantifying HA1–2-specific T-cell Responses
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Splenic lymphocytes were obtained from mice after the third vaccination, and the single-splenocyte suspensions were prepared for quantifying HA1–2-specific IFN-γ- or IL-4-producing cells using the BD ELISPOT set (BD Biosciences, Franklin Lakes, NJ, USA). All ELISPOT assays were performed according to our previously described procedure (11 (link)).
6
Quantification of HA1-2-specific IFN-γ/IL-4 Cells
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HA1-2-specific IFN-γ- or IL-4-producing cells were quantified in splenic lymphocyte cultures using the BD™ ELISPOT set (BD Biosciences) following the manufacturer’s protocol. Briefly, 96-well ELISPOT PVDF microplates were coated with a capture antibody in sterile PBS, incubated overnight at 4°C, and blocked (2 h at room temperature) with complete RPMI medium. Splenic lymphocytes were counted and plated in ELISPOT plates at 2 × 105 cells/well (in triplicate) in complete RPMI medium. Cells were mock stimulated with RPMI or stimulated with HA1-2 (5 μg/mL) for 24 h incubation at 37°C and 5% CO2. After the incubation period, ELISPOT plates were processed following the manufacturer’s specifications using a biotinylated antibody as a detection antibody, streptavidin-AKP (BD Pharmingen) and a BCIP/NBT Liquid Substrate System (Sigma-Aldrich) for assay development (20 min at room temperature). All assay plates were scanned and analyzed using an automated ELISPOT reader system (Bioreader 5000-Vβ, BioSys, Karben, Germany).
7
HA1-2 Vaccine Immune Responses
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Levels of HA1-2-specific IL-6 and IL-23 cytokines were assayed in splenocytes to evaluate cellular immune responses induced by HA1-2-fliC and PEI adjuvanted vaccines. Splenocytes (2 × 106 cells/mL) from immunized mice were incubated for 24 h at 37°C and 5% CO2 in complete RPMI medium with HA1-2 antigen (5 μg/mL). The culture supernatant was collected and concentrations of cytokines were quantitated by ELISA kits for IL-6 (BD Biosciences) and IL-23 (eBioscience), and the IL-6-producing cells were quantified in splenic lymphocyte cultures using the BD™ ELISPOT set (BD Biosciences) according to the manufacturer’s instructions.
8
ELISPOT Assay for B16 Antigen Detection
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ELISPOT assays were performed using the BD ELISPOT Set from BD Biosciences. Briefly, isolated splenocytes (5×105 cells/well) and B16 Ag or HBc87-95 peptide (10 μg/ml) were added to each well in triplicate and incubated at 37°C for 30 h. The spots were counted and analyzed with an ELISPOT Reader (Biosys, Germany).
9
Cytotoxic T Cell IFN-γ Response Assay
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The resulting CTLs were characterized for specific IFN-γ production in response to stimulation with target cells described above at weekly intervals, using the BD ELISPOT Set (BD Biosciences). CTLs were cultured with target cells at a ratio of 5:1 and incubated overnight. Spots were developed according to the instructions from the manufacturer and counted using an automated ImmunoSpot Analyzer (CTL-Cellular Technology Ltd) as previously described. 9 Each group was done in triplicates and the average was taken. The number of spots in each group was calculated by subtracting the number of spots in control wells (with CTL only) from the number of spots in each group (CTL and target cells). A positive response is defined as >200 spots/million cells and twice of the control. In antibody-blocking studies, the targets were incubated with 10 µg/mL of relevant mAb for 1 h at 37°C before adding the CTL. The antibodies used include anti-HLA-A, B, C mAb (W6/32), anti-HLA-DR mAb (L243), and mouse IgG2a kappa isotype control (BioLegend).
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