We used the mouse IFN gamma ELISPOT Ready-SET-Go!®; (eBioscience) to detect IFN-γ producing splenocytes. The procedure was performed according to the manufacturer‘s instructions. Briefly, ELISpot plates (MAIPS4510; Millipore) were coated with the capture antibody and incubated overnight at 4°C. The plates were washed twice with PBS and blocked for 1 h with R10 at rt. Splenocytes were incubated in the presence of 2 μg/mL pooled EDIII or EDI/II (negative control) peptides for 20 h at 37°C with 5% CO2. Unpulsed cells were used as controls for each group. After incubation, the plates were washed three times with PBS-T 0.05% and incubated for 2 h at rt with the biotinylated anti-IFN-γ antibody. Following another round of washes, the plates were incubated with avidin-HRP for 45 min at rt. Plates were washed three times with PBS-T 0.05% and the spots were developed with the “AEC substrate set” kit (BD biosciences). We used an automated stereomicroscope (KS ELISPOT, Zeiss, Oberkochem, Germany) to count the number of spots. The formula (# of spots in the pulsed well – # of spots in the unpulsed well) was used to calculate the number of IFN-γ producing cells/106 cells.
Ks elispot
Manufactured by Zeiss
Sourced in Germany
The KS ELISPOT is a piece of laboratory equipment designed for the detection and quantification of cells secreting specific proteins, known as cytokines or antibodies. The core function of the KS ELISPOT is to provide a sensitive and reliable method for the analysis of cellular immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Maips4510, by Merck Group (1 mentions)
Goat anti mouse igg hrp, by Jackson ImmunoResearch (1 mentions)
Mouse ifn γ elispot ready set go, by Thermo Fisher Scientific (1 mentions)
Ready set go kit, by Thermo Fisher Scientific (1 mentions)
Aec kit, by BD (1 mentions)
Anti ifn γ capture antibody, by BD (1 mentions)
Alkaline phosphatase conjugated streptavidin, by Merck Group (1 mentions)
Multiscreenhts plates, by Merck Group (1 mentions)
8 protocols using ks elispot
1
Mouse IFN-γ ELISPOT Assay
2
Antigen-Specific Memory B Cell Frequency
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The frequency of antigen-specific memory B cells was determined as previously described [48 (link)]. Briefly, the gp140 glycoprotein diluted in PBS (250 ng/well) was used to coat nitrocellulose plates overnight at rt. Antibody secreting cells (ASC) were detected using HRP goat anti-mouse IgG (Jackson Laboratories) and the reaction was developed by AEC colorimetric substrate. Number of spots per well was counted using an automated stereomicroscope (KS ELISpot, Zeiss).
3
IFN-γ ELISPOT Assay for Vaccine Response
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Splenocytes from immunized mice were assayed for their ability to secrete IFN-γ after in vitro stimulation with recombinant gp140 (5 μg/mL) using the ELISPOT assay. The ELISPOT assay was performed using mouse IFN-γ ELISPOT Ready-SET-Go! (eBiosciences) according to manufacturer’s instructions. Spots were counted using an automated stereomicroscope (KS ELISpot, Zeiss). The cutoff was 15 SFU per million splenocytes.
4
Dual Fluorescent EPISPOT Assay for CTCs
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In order to separate erythrocytes and leukocytes from CTCs, RosettSepTM reagent was added to 13–15 mL of EDTA blood (20 μL/mL) and processed following the manufacturer’s instructions (RosetteSepTM CTC Enrichment Cocktail Containing Anti-CD56, STEMCELL technologies). During the dual fluorescent PSA/FGF2-EPISPOT assay, harvested tumor cells were cultured for two days on a membrane coated with antibodies against PSA (5 ng/μl H-50; this antibody was given by Prof. Hans Lilja, Memorial Sloan Kettering Cancer Center, New-York) and FGF2 (10 ng/μl 500-M38, PeproTech). The antibodies should capture the secreted PSA/FGF2 proteins that are subsequently detected by secondary antibodies labeled with fluorochromes (5 ng/μl PSA-H117 labeled with AlexaFluor555; 0.9 ng/μl FGF2-biotin + anti-biotin-FITC). Single fluorescent PSA and FGF2 immunospots were counted under a fluorescent microscope using a video camera imaging and computer-assisted analysis (KS ELISPOT, Carl Zeiss Vision) as well as with the CTL Elispot reader.
5
ELISPOT Assay for IFN-γ Detection
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ELISPOT assays for the detection of IFN-γ producing splenocytes were performed using the Ready-SET-Go kit (eBioscience), according to the manufacturer’s instructions. Three hundred thousand splenocytes were incubated in the presence of 1 μg/mL of the recombinant MSP119 or MSP133 proteins. Control cells were left unpulsed. The AEC kit (BD biosciences) was used to develop the spots that were counted with the aid of an automated stereomicroscope (KS ELISPOT, Zeiss, Oberkochem, Germany). The number of IFN-γ producing cells/106 splenocytes was calculated after subtracting the number of cells in the unpulsed wells.
6
Enrichment and Detection of Circulating Melanoma Cells
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As CMCs are rare in peripheral blood, a pre-enrichment step was performed before the EPISPOT assay. To separate erythrocytes and leukocytes from CMCs, the RosettSepTM reagent (20 μL/mL) was added to 13–15 mL of blood collected in EDTA tubes, and enrichment was performed following the manufacturer’s instructions (RosetteSepTM CTC Enrichment Cocktail containing Anti-CD36, STEMCELL Technologies). During the S100-EPISPOT assay, enriched CMCs were cultured on a membrane coated with the anti-S100 8B10 antibody (10 ng/μL; Abcam) for 2 days. Then, secreted S100 captured by the 8B10 antibody was detected by incubation with another antibody against S100 (6G1: 3 ng/µL; Abcam) conjugated to AlexaFluor 488. Single fluorescent S100 immunospots were counted under a fluorescent microscope equipped with a camera and computer-assisted analysis (KS ELISPOT, Carl Zeiss Vision). The detailed procedure of the EPISPOT assay has been described by Soler et al. [21 (link)]. Results were corrected as “number of cells per 7.5 mL of blood” to be comparable with those obtained with the CellSearch system.
7
Cytotoxic T cell Activation Assay
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The ELISPOT plates were coated sterilely overnight with anti-IFN-γ capture antibody (BD Biosciences, San Jose, CA, USA) at 4°C. The plates were then washed once and blocked with AIM V medium containing 10% human serum for 2 h at room temperature. CD8-positive T cells separated from patients' PBMCs (5 × 103 cells/well), which were stimulated in vitro as described above, were then added to each well along with HLA-A24-transfected T2 (T2-A24) cells (5 × 104 cells/well) that had been preincubated with survivin-2B80-88 (10 μg/mL) or HIV with an HIV peptide as a negative control. After incubation in a 5% CO2 humidified chamber at 37°C for 24 h, the wells were washed vigorously five times with PBS and incubated with a biotinylated anti-human IFN-γ antibody (R&D Systems, Minneapolis, MN, USA) and HRP-conjugated avidin. Spots were visualized and analyzed using KS ELISPOT (Carl Zeiss, Jena, Germany).
8
B Cell Activation and Enumeration Assay
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Purified MZ B cells and FO B cells were cultured at 105 cells/200 µl complete RPMI medium as described in the Cell sorting, cell culture, and adoptive transfer section. After 48 h of culture, cells were washed and distributed at various concentrations into Multiscreen HTS plates (EMD Millipore) precoated with 10 µg/ml goat anti–mouse IgM (H + L; Southern Biotech) in 2% FBS RPMI 1640 medium, and then incubated for 24 h at 37°C in 5% CO2. Plates were washed, and then incubated with biotin-conjugated goat anti–mouse IgM (H + L; 1:5,000; Southern Biotechnology Associates) for 2 h at room temperature. Plates were washed, and then treated with alkaline phosphatase–conjugated Streptavidin (1:1,000; Sigma-Aldrich) and developed with 5-bromo-4-chloro-3-indoyl phosphate/NBT (Sigma-Aldrich). ISCs were enumerated using the KS ELISPOT (ZEISS).
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