ELISpot Human IFNγ (R&D Systems, USA) kit was used according to the manufacturer’s recommendations. Harvested CD8+ T cells were then plated and incubated at 37 °C for 24 h in the presence of irradiated peptide-pulsed PBMCs (40 Gy) that were used as stimulator cells. As a negative control, sorted CD8 T cells were incubated with irradiated non-pulsed PBMCs. Spots were revealed as mentioned in the manufacturer’s protocol and were counted using an ImmunoSpot S5 UV Analyzer (Cellular Technology Ltd., Shaker Heights, OH). IFNγ production was expressed as the number of peptide-specific spot-forming cells (SFC) per 106 CD8+ T cells after subtracting the spot counts from negative control wells.
Immunospot s5 uv analyzer
Manufactured by Cellular Technology
Sourced in United States
The ImmunoSpot S5 UV Analyzer is a laboratory equipment device designed for the detection and analysis of immune cells. It utilizes ultraviolet (UV) light to visualize and quantify cellular responses. The device provides accurate and objective measurements of cellular activities.
Automatically generated - may contain errors
Lab products found in correlation
Elispot human ifnγ, by R&D Systems (1 mentions)
Goat anti mouse igm, by Southern Biotech (1 mentions)
Goat anti mouse ig h l, by Southern Biotech (1 mentions)
Goat anti mouse igg, by Southern Biotech (1 mentions)
Bcip nbt plus substrate for elispot, by Mabtech (1 mentions)
Elispot kit, by R&D Systems (1 mentions)
4 protocols using immunospot s5 uv analyzer
1
Quantifying Peptide-Specific CD8+ T Cells
2
B Cell Spot-Forming Assay Protocol
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Filtration Plate MultiScreen HTS HA Sterile Plates (96 wells, MilliporeSigma MSHAS4510) were activated and coated for 1 hour at room temperature with goat anti–mouse Ig (H+L) (4 μg/mL; SouthernBiotech 1010-01, RRID:AB_2794121). After blocking overnight at 4°C, mouse splenocytes or peritoneal cells were plated in serial dilutions, starting with 105 cells per well, and incubated at 37°C in 5% CO2 atmosphere overnight. Spots of bound IgG or IgM were detected by adding the alkaline phosphatase–conjugated antibodies goat anti–mouse IgG (0.5 μg/mL; SouthernBiotech 1030-04, RRID:AB_2794293) or goat anti–mouse IgM (0.5 μg/mL; SouthernBiotech 1020-04, RRID:AB_2794200) for 1 hour at room temperature. The reaction was visualized by subsequent addition of 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium substrate (MilliporeSigma B5655-5TAB). The number of spots was assessed via a CTL ImmunoSpot S5 UV Analyzer equipped with ImmunoSpot ImmunoCapture and ImmunoSpot Counting softwares (Cellular Technology Ltd., RRID:SCR_011082).
3
Human IFN-γ ELISpot Assay for MDDC:CD4+ T Cell
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As previously described, human IFN-γ enzyme-linked immunosorbent spot (ELISpot) assays (Mabtech, USA) were carried out with MDDC:CD4+ T cell co-cultures as per the manufacturer's instructions [34 (link)]. After 16 h of ELISpot incubation, culture supernatants were gently aspirated and removed for additional experiments. Spots were developed using 1 µg/mL of biotinylated anti-IFN-γ antibody for 2 h, followed by 1 µg/mL of streptavidin-ALP for 1 h, a reaction that was stopped with 100 µl/well of “BCIP/NBT-plus substrate for ELISpot” (Mabtech, USA), then incubated for 15 min in the dark to develop the plate, with D-PBS washes in between. Spot forming units (SFU) were counted using an ImmunoSpot S5 UV Analyzer (Cellular Technology Ltd, USA) and ImmunoSpot 5.0.9 software. The raw SFU counts were corrected to 1 × 106 CD4+ T cells/mL and analyzed using GraphPad Prism version 8.0.1 (GraphPad Software, USA).
4
Granzyme B Secreting Cell Enumeration
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Granzyme B (GrB)-secreting cells were spotted and enumerated using ELISPOT kits and assay protocols obtained from R&D Systems (Minneapolis, MN). Briefly, 96-well PVDF membrane plates were coated overnight at 4°C with an anti-GrB capture mAb. Membranes were subsequently blocked for 2 hours and washed before erythrocyte-depleted splenocytes from T Ag-primed WT and IDO−/− mice were seeded at 3×105 cells/well and stimulated with indicated peptides. Concanavalin A (ConA) was used as a positive control at a final concentration of 5 µg/mL. The plates were incubated overnight at 37°C and 6% CO2 in a humidified atmosphere. Cells were then removed and the plates were washed before a biotin-labeled polyclonal Ab detecting mouse GrB was added to the wells. After overnight incubation at 4°C, the plates were washed and 100 µL of streptavidin-alkaline phosphatase (1∶60 in PBS/1% BSA) was added to each well. The plates were incubated at room temperature for an additional 2 hours and spots were visualized with BCIP/NBT (5-bromo-4-chloro-3′ indolylphosphate p-toluidine salt and nitro blue tetrazolium chloride in organic solvent) and subjected to automated evaluation using an ImmunoSpot S5 UV Analyzer (Cellular Technology Ltd., Cleveland, OH).
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