The peptide epitopes of EBOV GP and VP40 proteins that are specific for CD8+ T lymphocytes in BALB/c mouse (GP-T1: LYDRLASTV; GP-T2: GPCAGDFAF; VP40-T1: YFTFDLTALK; VP40-T2: TSPEKIQAIM) were selected according to previous reports (Warfield et al., 2005 (link); Wu et al., 2012 (link)), and synthesized (GeneScript, Nanjing, China). After the preparation of single cell suspensions from mouse splenocytes, ELISPOT assays were performed in pre-coated 96-well plates (Dakewe Biotech, China). The antibody-coated plates were blocked with complete RPMI 1640 medium for 2 h at room temperature. After blocking, 100 μl of splenocytes suspension (1 × 106 cells/ml) containing different peptides (10 μg/ml) were added to each well. A positive control PMA/ionomycin (Sigma) and a media negative control were included in all assays. The plates were incubated for 24 h in a humidified incubator at 37°C, 5% CO2. Plates were then washed and processed according to manufacturer’s instructions, and spots were enumerated using an ImmunoSpot reader and ImmunoSpot software (Cellular Technology Ltd.). Peptide-specific CD8+ T-cell frequency was expressed as Spots forming cells (SFCs)/1 × 105 splenocytes. Background spots (negative control wells) were subtracted from test wells. A positive response to a peptide was defined as having > 5 SFCs/1 × 105 splenocytes after subtraction of the background.
Immunospot software
Manufactured by Cellular Technology
Sourced in United States, Germany
The ImmunoSpot software is a digital analysis tool used for the quantification and characterization of immune cells. It provides an automated and standardized approach to analyze various types of immune assays, such as ELISPOT and other cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Immunospot s6 ultimate reader, by Cellular Technology (6 mentions)
Immunospot analyzer, by Cellular Technology (5 mentions)
Ionomycin, by Merck Group (5 mentions)
Immunospot reader, by Cellular Technology (4 mentions)
Immunospot 4.0 analyzer, by Cellular Technology (4 mentions)
Phorbol myristate acetate (pma), by Merck Group (4 mentions)
S6 ultra immunoscan reader, by Cellular Technology (4 mentions)
Ctl immunospot analyzer, by Cellular Technology (4 mentions)
Millipore elispot plates, by Merck Group (3 mentions)
Concanavalin a (cona), by Merck Group (3 mentions)
Bcip nbt, by Merck Group (3 mentions)
Mouse ifn γ single color elispot kit, by Cellular Technology (3 mentions)
Bovine calf serum (bcs), by Thermo Fisher Scientific (3 mentions)
Aminoethyl carbazole staining kit, by Merck Group (3 mentions)
Multiscreen 96 well filter plate, by Merck Group (3 mentions)
Elispot reagents, by Mabtech (3 mentions)
Anti cd3, by Thermo Fisher Scientific (3 mentions)
Tween 20, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Elispot plate, by Merck Group (3 mentions)
71 protocols using immunospot software
1
Identification of EBOV T Cell Epitopes
2
ZIKV-Specific Antibody-Secreting Cell Assay
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ELISpot assays were performed as previously described58 (link) with some modifications. Briefly, splenocytes were stimulated with 1 µg/ml R848 and 10 ng/ml recombinant human IL-2 (purchased from Mabtech In, OH). Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with ZIKV VLP (The Native Antigen Company, Oxford, UK, 15 mg/ml). To detect total IgG, the wells were coated with anti-human Ig capture Ab (Mabtech In). The stimulated splenocytes were harvested, washed, and added in duplicates to assess ZIKV-specific or total IgG ASCs. The plates were incubated overnight at 37 °C. This was followed by incubation with biotin-conjugated anti-mouse IgG (Mabtech In) was added for 2 h at room temperature, then 100 µL/well streptavidin-ALP were added for 1 h. Plates were developed with BCIP/NBT-Plus substrate until distinct spots emerge. The plates were washed with tap water and the plates were scanned using an ImmunoSpot 4.0 analyzer and the spots were counted with ImmunoSpot software (Cellular Technology Ltd., Cleveland, OH).
3
Analyzing Splenocyte Immune Responses
4
FMDV-specific IFN-γ ELISpot Assay
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FMDV-specific interferon γ (IFN-γ) SC frequencies were quantified to evaluate immune cell activation in splenocytes by porcine IFN-γ single-color enzymatic ELISpot assays (Cellular Technology Limited, USA). The plates were coated with 80 μL/well porcine IFN-γ capture solution at 4°C overnight. After washing with PBS one time, VLPs (1.0 and 10.0 μg/well) plus mitogen were plated in the wells (100 μL/well) and incubated in a 5% CO2 incubator for 20 min (performed in duplicate). After that, 100 μL of CTL-Test medium containing 3 × 105 PBMCs was added to each well and then incubated at 37°C in a 5% CO2 incubator for 24 h. The concanavalin A (50 μg/mL, 100 μL/well; Sigma–Aldrich, USA) and PBS were used as the positive and negative controls, respectively. After treatment with 80 μL/well of antiporcine IFN-γ detection solution at RT for 2 h, the wells were incubated with 80 μL/well of tertiary solution for 30 min at RT and then with the addition of 80 μL/well of blue developer solution in the dark for 15 min at RT. Spot numbers were determined with a camera and analyzed using ImmunoSpot Software (Cellular Technology Limited, USA). The number of spots in negative controls was subtracted from counts of spot-forming cells in stimulated wells.
5
SARS-CoV-2 Epitope-Specific T-cell Assay
6
Quantifying H1N1-Specific Memory B Cells
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H1N1-specific memory B cells (MBC) were measured from T2 by ELISPOT assay as described14 (link). For MBC enumeration, previously cryopreserved PBMC were thawed, rested, and stimulated with 5 µg/mL H1N1/09 vaccine antigen plus anti-CD28 mAb (1 µg/mL), CpG (as a positive control) or unstimulated (as negative control) for 5 days at 37 °C. On day 5, cells were plated in wells coated with goat anti-human IgG (2 µg/mL, Jackson Immunoresearch) at 100,000 cells/well for 4 hours at 37 °C and assayed for H1N1-specific IgG. Spots were read using Immunospot software and plate reader (Cellular Technologies LTD). H1N1-specific IgG results were normalized and graphed as proportion of the positive control.
7
ELISPOT Assay for Antigen-Specific Antibodies
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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.).
8
Quantifying Tetanus Toxoid-Specific Antibody-Secreting Cells
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TT-specific ASC were enumerated by ELISPOT, as previously described (15 (link), 19 (link), 20 (link), 38 (link)). MultiScreen High protein binding immobilon-P membrane plates (Millipore Corporation, Bedford, MA) were coated with 10 μg/ml TT overnight at 37°C, blocked with complete RPMI 1640 (Life Technologies BRL, Life Technologies, Paisley, U.K.). Duplicates of cells from spleen and bone marrow in four three-fold dilutions starting with 1 × 107 cells in 100 μL in complete RPMI 1640 per well were incubated for 5 hours at 37°C, washed and incubated with ALP-goat anti-mouse IgG (Southern Biotechnology Associates) overnight at 4°C, and developed by 5-bromo-4-chloro-3-indolylphosphate and NBT in AP development buffer (Bio-Rad Labs, Hercules, CA). The number of spots, each representing a cell secreting TT-specific IgG antibodies, was counted with ELISPOT reader ImmunoSpot® S6 ULTIMATE using ImmunoSpot® SOFTWARE (Cellular Technology Limited (CTL) Europe, Bonn, Germany).
9
ELISPOT Assay for PPS1 and TT-specific ASC
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PPS1- and TT-specific ASC were enumerated by ELISPOT, as previously described (9 (link), 48 (link)). MultiScreen High protein binding immobilon-P membrane plates (Millipore Corporation, Bedford, MA) were coated with 20 μg/ml PPS1 or 10 μg/ml TT overnight at 37°C, blocked with complete RPMI 1640 (Life Technologies BRL, Life Technologies, Paisley, U.K.). Duplicates of cells from spleen and BM were tested in four three-fold dilutions starting with 1 × 107 cells in 100 μL in complete RPMI 1640 per well (9 (link), 48 (link)) and incubated for 5 h at 37°C, washed and incubated with ALP-goat anti-mouse IgG (Southern Biotechnology Associates) overnight at 4°C, and developed by 5-bromo-4-chloro-3-indolylphosphate and NBT in AP development buffer (Bio-Rad Labs, Hercules, CA). The number of spots, each representing a cell secreting specific Abs, were counted by ELISPOT reader ImmunoSpot® S6 ULTIMATE using ImmunoSpot® SOFTWARE (Cellular Technology Limited (CTL) Europe, Bonn, Germany).
10
Quantifying HBV-specific T Cell Responses
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PBMC samples from the 65 patients with chronic HBV were thawed, washed in CTL Anti-AggregateTM medium (Cellular Technology Limited, CTL) and left overnight at 37°C in complete RPMI with 10% human serum. Cells were plated at 1E7/ml and overlapping peptide pool for the HBV-capsid protein (≫90% purity, JPT) was added to the culture. IL-2 at a final concentration of 2 IU/ml was added to the cells at day 2 and maintained until day 5. After a 5-day stimulation, quadruplicates of 2.5E5 expanded cells were re-stimulated with the same HBV-capsid peptide pool (or actin peptide pool as irrelevant peptide, when appropriate) in the presence of MEDI2790 or a control IgG isotype. Staphylococcal enterotoxin B was included as a positive control in every plate. HBV-specific T cell responses were quantified by ELISpot using ELISpotPLUS interferon (IFN)-γ pre-coated plates (MabTech) according to manufacturer’s instructions. Quantification was performed using the ImmunoSpot® reader and images were analyzed with the ImmunoSpot® software (Cellular Technology Limited, CTL).
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