ELISPOT assay was performed using Human IFN-γ ELISpotPRO kit (MABTECH, Cincinnati, OH) according to the manufacturer’s instruction. Briefly, antigen presenting cells were pulsed with respective peptides at 37° C for 20 h, and the residual peptides that did not bind to cells were washed out to prepare peptide-pulsed cells as the stimulator cells. T cells were pre-treated with IL-2 (35 U/mL) for 16 h and then co-incubated with peptide-pulsed stimulator cells (2 × 104 cells/well) at 37° C for 20 h in 96-well plate. The supernatant was transferred into a new 96-well plate for ELISA assay. Spots were captured and analyzed by an automated ELISPOT reader, ImmunoSPOT S4 (Cellular Technology Ltd, Shaker Heights, OH) and the ImmunoSpot Professional Software package, Version 5.1 (Cellular Technology Ltd). To measure the secreted cytokine levels in the supernatant, we used OptEIA Human IFN-γ ELISA set (BD Biosciences), Human Granzyme B ELISA development kit (MABTECH) and read absorbance in a microplate reader at 450/570 nm.
Immunospot s4
Manufactured by Cellular Technology
Sourced in United States
The ImmunoSPOT S4 is a multipurpose instrument designed for the analysis and quantification of secreted proteins, cytokines, and other analytes. It utilizes an Enzyme-Linked Immunospot (ELISPOT) assay technique to detect and measure the frequency of antigen-specific cells.
Automatically generated - may contain errors
Lab products found in correlation
Immunospot professional software package version 5, by Cellular Technology (3 mentions)
Human ifn γ elispotpro kit, by Mabtech (3 mentions)
Opteia human ifn γ elisa set, by BD (2 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Mabtech (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Human granzyme b elisa development kit, by Mabtech (1 mentions)
Multiscreen ip 96 well plates, by Merck Group (1 mentions)
Human ifn γ elispot kit, by BD (1 mentions)
Aec substrate set, by BD (1 mentions)
Elisa set, by BD (1 mentions)
Human serum, by Thermo Fisher Scientific (1 mentions)
Ifn γ elispot assay, by Mabtech (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
6 protocols using immunospot s4
1
ELISPOT Assay to Measure IFN-γ and Granzyme B
2
ELISPOT Assay for T Cell Response
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The human IFN-γ ELISPOT kit and AEC substrate set (BD Biosciences) were used to analyze the T cell response to the respective peptides. The ELISPOT assay was performed according to the manufacturer's instructions. Briefly, T2 or PSCCA0922 cells were pulsed with 20 µg/ml of the respective peptides at 37°C for 20 h, and the residual peptide that did not bind to cells was washed out to prepare peptide-pulsed cells as the stimulator cells. After removing 500 µl of supernatant from each well of in vitro CTL-inducing cultures, 200 µl of cell culture suspensions were harvested from each well and distributed to two new wells (100 µl each) on Multiscreen-IP 96 well plates (Millipore, Bedford, MA). The cells were co-incubated with peptide-pulsed cells (1×104 cells/well) at 37°C for 20 h. HIV peptide-pulsed cells were used as a negative control. Spots were captured and analyzed by an automated ELISPOT reader, ImmunoSPOT S4 (Cellular Technology Ltd, Shaker Heights, OH) and the ImmunoSpot Professional Software package, Version 5.0 (Cellular Technology Ltd).
3
IFNγ Secretion Assay of TILs
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IFNγ secretion of TILs was measured by ELISPOT using Human IFNγ ELISpotPRO kit (MABTECH, Catalog number 3420–2APW-10) following the manufacturer’s instruction. Briefly, 5 × 104 resting TILs were co-cultured with 2 × 104 peptide-pulsed C1R A02:01 cells at 37°C for 20 hours in a 96-well plate. Spots were captured and analyzed by an automated ELISPOT reader, ImmunoSPOT S4 (Cellular Technology Ltd, Shaker Heights, OH), and analyzed with the ImmunoSpot Professional Software package, Version 5.1 (Cellular Technology Ltd).
The amount of secreted IFNγ, IL-2, and TNFα in the supernatant of T cells were quantified with ELISA set (BD Biosciences, Catalog number 555142, 555190, 555212). Briefly, 5 × 104 resting KIAA1429D1358E TCR-T cells were co-cultured with 2 × 104 peptide-pulsed C1R A02:01 cells at 37°C for 20 hours in a 96-well plate. The supernatant was collected, and the concentration of each protein was measured according to the manufacturer’s instruction.
The amount of secreted IFNγ, IL-2, and TNFα in the supernatant of T cells were quantified with ELISA set (BD Biosciences, Catalog number 555142, 555190, 555212). Briefly, 5 × 104 resting KIAA1429D1358E TCR-T cells were co-cultured with 2 × 104 peptide-pulsed C1R A02:01 cells at 37°C for 20 hours in a 96-well plate. The supernatant was collected, and the concentration of each protein was measured according to the manufacturer’s instruction.
4
Quantifying Antigen-Specific CTL Responses
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The antigen specific CTL response was determined by interferon-γ (IFN-γ) ELISpot assay according to the manufacturer’s protocol (Mabtech, Stockholm, Sweden). Briefly, 96-well plates with nitrocellulose membranes (Millipore, Bedford, MA) were pre-coated with primary anti-IFN-γ antibody (1-D1K) at 4 °C overnight. The plates were blocked with AIM-V medium containing 5% human serum (Invitrogen). Target cells (2 × 104/well) and CTL clones (2 × 103/well) were co-cultured in 200 µL of culture medium for 24 h in triplicate. These wells were treated with biotinylated secondary anti-IFN-γ antibody (7-B6-1), followed by incubation with HRP-reagent and stained with TMB (Mabtech). The spots were then quantified with automated ELISpot reader, ImmunoSPOT S4 (Cellular Technology Ltd, Cleveland, OH). Positive CTL responding specifically to the vaccinated peptide were evaluated and classified according to a previously described algorithm [29 (link)].
5
IFN-γ ELISpot Assay for CTL Response
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IFN-γ ELISpot assay was performed using a commercial kit (Mabtech, Stockholm, Sweden) to determine the CTL response [37, (link)38] (link). Briefly, 96-well plates with nitrocellulose membranes (Merck KGaA) were pre-coated with the primary anti-IFN-γ antibody at 4 °C overnight. After blocking with AIM-V medium containing 5% human serum, target cells (2 × 10 4 /well) and KIF20A peptidespecific CTL clones (2 × 10 3 /well) were co-cultured in 200 µL of culture medium at 37 °C for 24 h. These wells were treated with biotinylated secondary anti-IFN-γ mAb, followed by incubation with HRP-reagent and stained with TMB (Mabtech). The spots were then quantified with ImmunoSPOT S4 (Cellular Technology Ltd., Cleveland, OH, USA).
6
T Cell Functional Assays
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Interferon (IFN)-γ secretion of T cells were detected by ELISPOT using Human IFN-γ ELISpotPRO kit (MABTECH, Catalog number 3420-2APW-10) according to the manufacturer’s instruction. Briefly, APCs (C1R-A24 or C1R-A02 cells) were pulsed with each respective peptide before co-culture for 16 h at 37°C, 5% CO2. T cells were pre-treated with IL-2 (35 U/mL) for 16 h and then co-cultured with the peptide-pulsed APCs (2 × 104 APCs and 5 × 104 engineered T cells/well) at 37°C for 20 h in 96-well plate. Spots were captured and analyzed by an automated ELISPOT reader, ImmunoSPOT S4 (Cellular Technology Ltd, Shaker Heights, OH) and the ImmunoSpot Professional Software package, Version 5.1 (Cellular Technology Ltd).
OptEIA Human IFN-γ ELISA set, OptEIA Human IL2 ELISA set, OptEIA Human TNFα ELISA set (BD Biosciences, Catalog number 555142, 555190, 555212) were used to measure the secreted IFN-γ, IL2, and TNFα levels in the supernatant of co-cultured engineered T cells and APCs. Briefly, APCs were pulsed with respective peptides at 37°C for 16 h and 5% CO2. T cells were pre-treated with IL-2 (35 U/mL) for 16 h and then cocultured with the peptide-pulsed APCs (2 × 104 cells/well) at 37°C for 20 h in a 96-well plate. The supernatant was transferred into another 96-well plate, and the concentration of each protein was measured according to the manufacturer’s instruction.
OptEIA Human IFN-γ ELISA set, OptEIA Human IL2 ELISA set, OptEIA Human TNFα ELISA set (BD Biosciences, Catalog number 555142, 555190, 555212) were used to measure the secreted IFN-γ, IL2, and TNFα levels in the supernatant of co-cultured engineered T cells and APCs. Briefly, APCs were pulsed with respective peptides at 37°C for 16 h and 5% CO2. T cells were pre-treated with IL-2 (35 U/mL) for 16 h and then cocultured with the peptide-pulsed APCs (2 × 104 cells/well) at 37°C for 20 h in a 96-well plate. The supernatant was transferred into another 96-well plate, and the concentration of each protein was measured according to the manufacturer’s instruction.
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