The presence of Ag-specific T cells in the spleens of immunized animals was measured by IFNγ production in response to restimulation with corresponding immunodominant peptides by standard ELISPOT assay (Cellular Technology, Shaker Heights, OH, USA) according to the manufacturer’s protocol. Splenocytes were used in the assay either directly after harvesting or after they were stimulated with immune peptides and cultured for 7 days in the presence of 10 ng/mL interleukin-21 (IL-21) and 25 ng/mL IL-15. For all experiments, splenocytes were seeded at a concentration of 2 × 105 cells/well and stimulated with a single peptide or a pool of peptides of the corresponding Ag at a final concentration of 1 μg/mL. The peptides used in the study were the H-2Db immunodominant peptide mgp10025-33 EGSRNQDWL (Anaspec), H-2Db-restricted epitope mTRP1455-463 TAPDNLGYA (GenScript), and H-2Kb-restricted epitope mTRP2180-188 SVYDFFVWL (GenScript). For tyrosinase, the pool of overlapping peptides covering the full sequence of human tyrosinase was used (PepMix, JPT Peptide Technologies). Plates were cultured for 24 h and developed according to the manufacturer’s instructions. The IFNγ-secreting spots were counted with an ELISPOT reader. The results are presented as the number of spots per 1 × 106 cells.
Elispot assay
Manufactured by Cellular Technology
Sourced in United States
The ELISPOT assay is a laboratory technique used to detect and quantify the secretion of specific proteins, such as cytokines, by individual cells. The assay involves culturing cells in a specialized plate coated with antibodies that capture the protein of interest as it is secreted by the cells. The captured proteins are then visualized using a detection system, allowing for the identification and enumeration of the individual protein-secreting cells.
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4 protocols using elispot assay
1
Quantifying Ag-specific T Cell Responses
2
ELISpot Assay for Mouse Splenocyte Cytokine Response
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For ELISpot, splenocytes were isolated and resuspended with CTL-Test™ Medium containing CTL Serum-free Media with 1% Penicillin-Streptomycin (Gibco, Cat# 15140122) and 1% fresh L-glutamine (Beyotime Biotechnology, Cat# C0212). IL-2- and IL-5-positive splenocytes was performed according to the manufacturer's instructions for the ELISpot assay (Cellular Technology Ltd, USA). Briefly, 96-well plates were precoated with both anti-mouse IL-2 and IL-5 monoclonal antibodies. 3 × 105 cells of isolated mouse splenocytes per well were stimulated at 37 °C for 48 h using 1 μg/mL Spike trimer protein (ACRO, Cat # SPN-C52Hz). Wash plates two times with PBS and then two times with 0.05% Tween-PBS, detection antibodies were added and incubated at room temperature for 2 h. After three times washing, Tertiary Solution were added and incubated at room temperature for 30 min. Finally, the plates were washed two times with 0.05% Tween-PBS and then two times with distilled water. Add Blue Developer Solution and incubate at room temperature for 15 min. The spots were counted under CTL ImmunoSpot® Analyzers (Cellular Technology Ltd, USA).
3
ELISpot Assay for Measuring TRP2 and IFN-γ T Cells
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The amount of SVYDFFVWL (TRP2(180–188) and adenovirus-specific, activated, IFN-γ-secreting T cells was measured by ELISpot assay (Cellular Technology, OH, USA), according to the manufacturer’s instructions. Briefly, 2 μg of SVYDFFVWL peptide was used to stimulate the APCs (NB; this peptide contained only the MHC class I epitope in order to be able to rule out any unspecific stimulation, which could derive from the polylysine sequence used in the PeptiCRAd platform). After 3 days of stimulation, plates were stained and sent to Cellular Technology-Europe for counting of the spots.
4
Measuring Antigen-Specific T Cell Responses
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For analysis of Ova-specific T cells, IFNγ response to re-stimulation of splenocytes with Ova-immunodominant peptides (AnaSpec) MHC class I Ova257–264 and MHC class II Ova323–339 was measured by standard ELISPOT assay (Cellular Technology, Shaker Heights, OH, USA) according to the manufacturer’s protocol. For analysis of Tyr- and gp100-specific T cells, IFNγ and TNF-α were measured in re-stimulated splenocytes by the double-color mouse IFNγ/TNFα ImmnunoSpot system (Cellular Technology, Shaker Heights, OH, USA). Splenocytes from the mice immunized with 663-optTyr were re-stimulated with the pool of human Tyr peptides (JPT Peptide Technologies). Splenocytes from 663-opt-gp100-immunized mice were re-stimulated with mouse gp100 peptide epitope mGP10025–33 EGSRNQDWL (AnaSpec). For all experiments, splenocytes were seeded at 5 × 105 cells per well and were stimulated with corresponding peptides at a final concentration of 5 μg/mL. Plates were cultured for 24 h and then developed according to the manufacturer’s instructions. The IFNγ- and TNF-α-secreting spots were counted with an ELISPOT reader. Results are presented as number of spots per 106 cells.
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